Agents that bind a target in pulmonary tissue for targeting respiratory diseases

ABSTRACT

Disclosed is the use of an agent (e.g., antibody fragment, antagonist, ligand, dAb monomer) that binds a target in pulmonary tissue for the manufacture of a long action or long therapeutic window formulation for local delivery to pulmonary tissue, and methods for administering an agent that binds a target in pulmonary tissue to a subject to produce a long therapeutic window in pulmonary tissue. The formulation is for, and the method comprises, administering locally to pulmonary tissue. Also disclosed is the use of antagonists of TNFR1 for the manufacture of a formulation or medicament for treating, preventing or suppressing lung inflammation or a respiratory disease, and methods of treating such diseases. Also disclosed are the use of agents a for the manufacture of a delivery device (e.g., inhaler, intranasal delivery device) for the treatment or prevention of lung inflammation or a respiratory disease, and a delivery device for the treatment or prevention of lung inflammation or a respiratory disease that contains an agent as described herein.

RELATED APPLICATIONS

This application is the U.S. National Stage of International ApplicationNo. PCT/GB2006/003935, filed Oct. 23, 2006, published in English andclaims priority under 35 U.S.C. §119 or 365 to United Kingdom,Application No. GB 0521621.3, filed Oct. 24, 2005. The entire teachingsof the above application are incorporated herein by reference.

BACKGROUND OF THE INVENTION

The in vivo use of many agents with therapeutic or diagnostic potentialis not possible. Larger agents that have in vivo serum half-lives thatare sufficiently long to allow for therapeutic or diagnostic efficacyoften are unable to penetrate tissues or organs to produces a desiredtherapeutic or diagnostic effect at a desired location. Smaller agentsare able to enter tissues and organs, but frequently have short in vivoserum half-lives, and are rapidly cleared from the systemic circulation.For example, the in vivo serum half-life of dAb monomers is about 30minutes. (See, Examples 9 and 1.3 of WO 2004/081026 A2.) Similarly, thein vivo serum half-life of antigen-binding fragments of antibodies,particularly Fv fragments, is also short and makes them unsuitable formany in vivo therapeutic and diagnostic applications. (Peters et al,Science 286(5439):434 (1999).) Further, altering or modifying suchagents to increase the in vivo serum half-life can reduce the activityof the agent.

A need exists for methods for administering agents (e.g., to pulmonarytissue) to produce a long therapeutic window for the agent.

Agents that bind TNF and neutralize its activity have proven to beeffective therapeutic agents for certain inflammatory conditions, suchas arthritis. However, agents that bind TNF have not been demonstratedto be effective in treating lung inflammation or respiratory diseases,such as chronic obstructive pulmonary disease (COPD). (See, e.g., vander Vaart et al., Am. J. Respir. Crit. Care Med., 172(4):465-9 (2005),Rennard et al., Proc. Amer. Thorac. Soc., 2(Abstract Issue):A133, A541(2005), Abdelhady et al., Proc. Amer. Thorac. Soc., 2(AbstractIssue):A133 (2005).) Moreover, therapeutic agents that target TNF alpha,such as ENBREL® (etanercept; Immunex Corporation) antagonize TNFR1 andTNFR2, and administering such agents can produce immunosuppression andrelated side effects (e.g., serious infections). These side effects canlimit the use of such agents, particularly for chronic diseases wherethe agent is administered over a long period. (Kollias G. andKontoyiannis D., Cytokine Growth Factor Rev., 13(4-5):315-321 (2002).)In contrast, agents that specifically antagonize TNFR1 would havereduced side effects. However, targeting TNFR1 is difficult becauseagents that cause the receptor to cluster can activate signaling throughthe receptor, which can lead to the elaboration of inflammatorymediators such as TNF. In fact, multivalent agents that bind TNFR1, suchas anti-TNFR1 antibodies, can induce TNFR1 clustering and signaltransduction in the absence of TNF and are commonly used as TNFR1agonists. (See, e.g., Belka et al., EMBO, 14(6):1156-1165 (1995);Mandik-Nayak et al., J. Immunol, 167:1920-1928 (2001).) Accordingly,multivalent agents that bind TNFR1, are generally not effectiveantagonists of TNFR1 even if they block the binding of TNFα to TNFR1.

A need exists for improved agents that antagonize TNF and method foradministering such agents to treat lung inflammation and lung disease.

SUMMARY OF THE INVENTION

The invention relates to use of an agent that binds a target inpulmonary tissue (e.g., antibody fragment, antagonist, ligand, dAbmonomer) for the manufacture of a long action or long therapeutic windowformulation for local administration to pulmonary tissue, or for themanufacture of a medicament for local administration to pulmonary tissueof a low dose effective amount of said agent, wherein at least 50% ofthe pulmonary tissue level of agent is maintained for a period of atleast about 4 hours. Preferably, the formulation or medicament is forlocal administration to the lung. Preferably, a lung level of at leastabout 1% of the amount of agent in the formulation or medicament ismaintained for at least 4 hours after local administration. Morepreferably, the agent does not substantially enter the systemiccirculation. In some embodiments, the agent has an in vivo serum halflife of about 1 second to about 12 hours. In other embodiments, theformulation or medicament is for administering a dose of no more thanabout 10 mg/kg/day.

The invention relates to use of a domain antibody (dAb) that binds atarget in pulmonary tissue for the manufacture of a daily doseformulation for local administration to pulmonary tissue, wherein atleast 50% of the lung level of agent is maintained for a period of atleast about 4 hours.

The invention relates to use of a domain antibody (dAb) that binds atarget in pulmonary tissue for the manufacture of a formulation fortreatment or prevention of a respiratory disease, wherein theformulation is for local administration to pulmonary tissue, and doesnot substantially enter the systemic circulation. In one embodiment, upto about 10 mg of a dAb that binds a target in pulmonary tissue is used.Preferably, the target in pulmonary tissue mediates lung inflammation ora pulmonary disease.

The invention relates to an inhaler or intranasal delivery device forproviding a metered dose of a domain antibody (dAb) formulation to asubject for the treatment or prevention of a respiratory disease orcondition, wherein the inhaler or intranasal delivery device comprises adAb formulation and provides a metered daily dose containing up to 10 mgof cab. The invention also relates to use of a domain antibody (dAb)formulation in the manufacture of an inhaler or intranasal deliverydevice, for the purpose of providing a long-acting inhaled dAbformulation for local delivery to the lung.

The invention also relates to a method for administering an agent thatbinds a target in pulmonary tissue to a subject to produce a longtherapeutic window in pulmonary tissue, comprising administering locallyto pulmonary tissue of said subject an effective amount of said agent.

The invention also relates to a method for administering an agent thatbinds a target in pulmonary tissue to a subject to produce a longtherapeutic window in pulmonary tissue, comprising selecting an agentthat has an in vivo serum half-life of about 1 second to about 12 hoursand binds a target in pulmonary tissue, and administering locally topulmonary tissue of said subject an effective amount of said agent.

Suitable agents for use in the invention can bind a target in pulmonarytissue selected from the group consisting of TNFR1, IL-1, IL-1R, IL-4,IL-4R, IL-5, IL-6, IL-6R, IL-8, IL-8R, IL-9, IL-9R, IL-10, IL-12 IL-12R,IL-13, IL-13Rα1, IL-13Rα2, IL-15, IL-15R, IL-16, IL-17R, IL-17, IL-18,IL-18R, IL-23 IL-23R, IL-25, CD2, CD4, CD11a, CD23, CD25, CD27, CD28,CD30, CD40, CD40L, CD56, CD138, ALK5, EGFR, FcER1, TGFb, CCL2, CCL18,CEA, CR8, CTGF, CXCL12 (SDF-1), chymase, FGF, Furin, Endothelin-1,Eotaxins (e.g., Eotaxin, Eotaxin-2, Botaxin-3), GM-CSF, ICAM-1, ICOS,IgE, IFNa, I-309, integrins, L-selectin, MIF, MIP4, MDC, MCP-1, MMPs,neutrophil elastase, osteopontin, OX-40, PARC, PD-1, RANTES, SCF, SDF-1,siglec8, TARC, TGFb, Thrombin, Tim-1, TNF, TNFR1, TRANCE, Tryptase,VEGF, VLA-4, VCAM, α4β7, CCR2, CCR3, CCR4, CCR5, CCR7, CCR8,alphavbeta6, alphavbeta 8, cMET, and CD8.

In an embodiment, an agent for use in the invention can bind a targetselected from the group consisting of a protein in the TNF signallingcascade. Preferably, this protein target is selected from the groupcomprising TNF alpha, TNF beta, TNFR2, TRADD, FADD, Caspase-8, TNFreceptor-associated factor (TRAF), TRAF2, receptor-interacting protein(RIP), Hsp90, Cdc37, IKK alpha, IKK beta, NEMO, inhibitor of kB (IkB),NF-kB, NF-kB essential modulator, apoptosis signal-regulated kinase-1(aSMase), neutral sphingomyelinase (nSMase), ASK1, Cathepsin-B, germinalcenter kinase (GSK), GSK-3, factor-associated death domain protein(FADD), factor associated with neutral sphingomyelinase activation(FAN), FLIP, JunD, inhibitor of NF-kB kinase (IKK), MKK3, MKK4, MKK7,IKK gamma, mitogen-activated protein kinase/Erk kinase kinase (MEKK),MEKK1, MEKK3, NIK, poly(ADP-ribose) polymerase (PARP), PKC-zeta, RelA,T2K, TRAF1, TRAF5, death effector domain (DED), death domain (DD), deathinducing signalling complex (DISC), inhibitor of apoptosis protein(IAP), c-Jun N-erminal kinase (JNK), mitogen-activated protein kinase(MAPK), phosphoinositide-3OH kinase (PI3K), protein kinase A (PKA), PKB,PKC, PLAD, PTEN, rel homology domain (RHD), really interesting new gene(RING), stress-activated protein kinase (SAPK), TNF alpha-convertingenzyme (TACE), silencer of death domain protein (SODD), andTRAF-associated NF-kB activator (TANK). With regard to these preferredtargets, reference is made to WO04046189, WO04046186 and WO04046185(incorporated herein by reference) which provide guidance on theselection of antibody single variable domains for targetingintracellular targets.

The invention relates to use of an antagonist of TNFR1 (e.g., ligand,dAb monomer) for use in the manufacture of a medicament for treating,suppressing or preventing lung inflammation and/or a respiratorydisease.

The invention also relates to methods for treating, suppressing orpreventing lung inflammation and/or a respiratory disease comprising,selecting an antagonist of Tumor Necrosis Factor Receptor 1 (TNFR1) thathas efficacy in a suitable animal model of respiratory disease whenadministered in an amount that does not exceed about 10 mg/kg/day,wherein efficacy in said animal model exists when cellular infiltrationof the lungs, as assessed by total cell count in bronchoalveolar lavage,is inhibited relative to untreated control with p≦0.05; andadministering (e.g., locally administering to pulmonary tissue) aneffective amount of said antagonist of TNFR1 to a subject in needthereof.

Respiratory diseases that can be treated, suppressed or prevented usingthe medicaments, formulations and methods of the invention include lunginflammation, chronic obstructive pulmonary disease, asthma, pneumonia,hypersensitivity pneumonitis, pulmonary infiltrate with eosinophilia,environmental lung disease, pneumonia, bronchiectasis, cystic fibrosis,interstitial lung disease, primary pulmonary hypertension, pulmonarythromboembolism, disorders of the pleura, disorders of the mediastinum,disorders of the diaphragm, hyperventilation, hyperventilation, sleepapnea, acute respiratory distress syndrome, mesothelioma, sarcoma, graftrejection, graft versus host disease, lung cancer, allergic rhinitis,allergy, asbestosis, aspergilloma, aspergillosis, bronchiectasis,chronic bronchitis, emphysema, eosinophilic pneumonia, idiopathicpulmonary fibrosis, invasive pneumococcal disease, influenza,nontuberculous mycobacteria, pleural effusion, pneumoconiosis,pneumocytosis, pneumonia, pulmonary actinomycosis, pulmonary alveolarproteinosis, pulmonary anthrax, pulmonary edema, pulmonary embolus,pulmonary inflammation, pulmonary histiocytosis X, pulmonaryhypertension, pulmonary nocardiosis, pulmonary tuberculosis, pulmonaryveno-occlusive disease, rheumatoid lung disease, sarcoidosis, Wegener'sgranulomatosis, and non-small cell lung carcinoma.

The invention also relates to the ligands and dAbs described herein.

The invention also relates to a ligand comprising a protein moiety thathas a binding site with binding specificity for TNFR1, wherein saidprotein moiety comprises an amino acid sequence that is the same as theamino acid sequence of CDR3 of an anti-TNFR1 dAb disclosed herein.

In some embodiments, the ligand comprising a protein moiety that has abinding site with binding specificity for TNFR1, wherein the proteinmoiety has an amino acid sequence that is the same as the amino acidsequence of CDR3 of an anti-TNFR1 dAb disclosed herein, and alsocomprises an amino acid sequence that is the same as the amino acidsequence of CDR1 and/or CDR2 of an anti-TNFR1 dAb disclosed herein.

In other embodiments, the ligand comprises a immunoglobulin singlevariable domain that binds TNFR1, wherein the immunoglobulin singlevariable domain that binds TNFR1 differs from the amino acid sequence ofall anti-TNFR1 dAb disclosed herein at no more than 25 amino acidpositions and has a CDR1 sequence that has at least 50% identity to theCDR1 sequence of the anti-TNFR1 dAbs disclosed herein.

In other embodiments, the ligand comprises an immunoglobulin singlevariable domain that binds TNFR1, wherein the amino acid sequence of theimmunoglobulin single variable domain that binds TNFR1 differs from theamino acid sequence of an anti-TNFR1 dAb disclosed herein at no morethan 25 amino acid positions and has a CDR2 sequence that has at least50% identity to the CDR2 sequence of the anti-TNFR1 dAbs disclosedherein.

In other embodiments, the ligand comprises an immunoglobulin singlevariable domain that binds TNFR1, wherein the amino acid sequence of theimmuoglobulin single variable domain that binds TNFR1 differs from theamino acid sequence of an anti-TNFR1 dAb disclosed herein at no morethan 25 amino acid positions and has a CDR3 sequence that has at least50% identity to the CDR3 sequence of the anti-TNFR1 dAbs disclosedherein.

In other embodiments, the ligand comprises an immunoglobulin singlevariable domain that binds TNFR1, wherein the amino acid sequence of theimmunoglobulin single variable domain that binds TNFR1 differs from theamino acid sequence of an anti-TNFR1 dAb disclosed herein at no morethan 25 amino acid positions and has a CDR1 sequence and a CDR2 sequencethat has at least 50% identity to the CDR1 or CDR2 sequences,respectively, of the anti-TNFR1 dAbs disclosed herein.

In other embodiments, the ligand comprises an immunoglobulin singlevariable domain that binds TNFR1, wherein the amino acid sequence of theimmunoglobulin single variable domain that binds TNFR1 differs from theamino acid sequence of an anti-TNFR1 dAb disclosed herein at no morethan 25 amino acid positions and has a CDR2 sequence and a CDR3 sequencethat has at least 50% identity to the CDR2 or CDR3 sequences,respectively, of the anti-TNFR1 dAbs disclosed herein.

In other embodiments, the ligand comprises an immunoglobulin singlevariable domain that binds TNFR1, wherein the amino acid sequence of theimmunoglobulin single variable domain that binds TNFR1 differs from theamino acid sequence of an anti-TNFR1 dAb disclosed herein at no morethan 25 amino acid positions and has a CDR1 sequence and a CDR3 sequencethat has at least 50% identity to the CDR1 or CDR3 sequences,respectively, of the anti-TNFR1 dAbs disclosed herein.

In other embodiments, the ligand comprises an immunoglobulin singlevariable domain that binds TNFR1, wherein the amino acid sequence of theimmunoglobulin single variable domain that binds TNFR1 differs from theamino acid sequence of an anti-TNFR1 dAb disclosed herein at no morethan 25 amino acid positions and has a CDR1 sequence, a CDR2 sequenceand a CDR3 sequence that has at least 50% identity to the CDR1, CDR2 orCDR3 sequences, respectively, of the anti-TNFR1 dAbs disclosed herein.

In another embodiment, the invention is a ligand comprising animmunoglobulin single variable domain that binds TNFR1, wherein theimmunoglobulin single variable domain that binds TNFR1 has a CDR1sequence that has at least 50% identity to the CDR1 sequences of ananti-TNFR1 dAb disclosed herein.

In another embodiment, the invention is a ligand comprising animmunoglobulin single variable domain that binds TNFR1, wherein theimmunoglobulin single variable domain that binds TNFR1 has a CDR2sequence that has at least 50% identity to the CDR2 sequences of ananti-TNFR1 dAb disclosed herein.

In another embodiment, the invention is a ligand comprising animmunoglobulin single variable domain that binds TNFR1, wherein theimmunoglobulin single variable domain that hinds TNFR1 has a CDR3sequence that has at least 50% identity to the CDR3 sequences of ananti-TNFR1 dAb disclosed herein.

In another embodiment, the invention is a ligand comprising allimmunoglobulin single variable domain that binds TNFR1, wherein theimmuoglobulin single variable domain that binds TNFR1 has a CDR1 and aCDR12 sequence that has at least 50% identity to the CDR1 and CDR2sequences, respectively, of an anti-TNFR1 dAb disclosed herein.

In another embodiment, the invention is a ligand comprising animmunoglobulin single variable domain that binds TNFR1, wherein theimmunoglobulin single variable domain that binds TNFR1 has a CDR2 and aCDR3 sequence that has at least 50% identity to the CDR2 and CDR3sequences, respectively, of an anti-TNFR1 dAb disclosed herein.

In another embodiment, the invention is a ligand comprising animmunoglobulin single variable domain that binds TNFR1, wherein theimmunoglobulin single variable domain that binds TNFR1 has a CDR1 and aCDR3 sequence that has at least 50% identity to the CDR1 and CDR3sequences, respectively, of an anti-TNFR1 dAb disclosed herein.

In another embodiment, the invention is a ligand comprising animmunoglobulin single variable domain that binds TNFR1, wherein theimmunoglobulin single variable domain that binds TNFR1 has a CDR1, CDR2,and a CDR3 sequence that has at least 50% identity to the CDR1, CDR2 andCDR3 sequences, respectively, of an anti-TNFR1 dAb disclosed herein.

The invention also relates to an isolated or recombinant nucleic acidencoding any of the ligands of the invention. In other embodiments, theinvention relates to a vector comprising the recombinant nucleic acid ofthe invention.

The invention also relates to a host cell comprising the recombinantnucleic acid of the invention or the vector of the invention.

The invention also relates to a method for producing a ligand,comprising maintaining a host cell of the invention under conditionssuitable for expression of a nucleic acid or vector of the invention,whereby a ligand is produced. In other embodiments, the method ofproducing a ligand further comprises isolating the ligand.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a plot showing that an antagonist of TNFR1 has superiorefficacy in comparison to other therapeutic agents when administeredlocally to pulmonary tissue in a subchronic model of tobaccosmoke-induced (TS) chronic obstructive pulmonary disease (COPD) inC57BL/6 mice. The plot shows the number of cells present inbronchoalveolar lavage (BAL) of mice at completion of the studydescribed in Example 1. The individual data points for each mouse in thestudy and the group averages (means; horizontal lines) are shown. Theresults show that anti-TNFR1 dAb monomer (Dom1) locally administered tothe lung by intranasal administration reduced the number of cells in BALby 72% compared to the untreated group. The results also show that localadministration to the lung of a therapeutic agent that targets TNF(ENBREL® (etanercept; Immunex Corporation)) did not have a statisticallysignificant effect on the number of cells in BAL. The results furthershow that anti-TNFR1, dAb monomer (Dom1) locally administered to thelung by intranasal administration was more effective in reducing thenumber of cells in BAL that a phosphodiesterase 4 inhibitor (PDE4I, BAY19-8004) that was administered at a high dose of 10 mg/kg orally twice aday (b.i.d.). TS, tobacco smoke-induced; Veh, vehicle; ns, notstatistically significant.

FIG. 2 is a plot showing that an antagonist of TNFR1 has superiorefficacy in comparison to a therapeutic agent that targets TNF whenadministered systemically in a subchronic model of tobacco smoke-induced(TS) chronic obstructive pulmonary disease (COPD) in C57BL/6 mice. Theplot shows the number of cells present in BAL of mice at completion ofthe study described in Example 2. The individual data points for eachmouse in the study and the group averages (horizontal lines) are shown.The results show that PEGylated anti-TNFR1 dAb monomer (TNFR1)systemically administered by intraperitoneal administration reduced thenumber of cells in BAL by 60% compared to the untreated group. Theresults also show that systemic administration of a therapeutic agentthat targets TNF (ENBREL® (etanercept; Immunex Corporation)) resulted ina 12% increase in the number of cells in BAL, although this increase wasnot statistically significant. TS, tobacco smoke-induced; Veh, vehicle;ns, not statistically significant; i.p. intraperitoneal.

FIG. 3 is a histogram in which the data for certain groups that areshown in FIGS. 1 and 2 are replotted along with the results for a studyin which an oral steroid (Dexamethasone) was administered in the model.The histogram shows that local administration of anti-TNFR1 dAb monomer(DOM/ADS101-native (Dom1 in FIG. 1)) to the lung by intranasaladministration (1 mg/kg administered once each day (q.d.)), and systemicadministration of PEGylated anti-TNFR1 dAb monomer (DOM/ADS101-pegylated(TNFR1 in FIG. 2)) by intraperitoneal administration (10 mg/kgadministered once every two days (q.a.d.)) were more efficacious in themodel than phospliodiesterase 4 inhibitor (PDE4I) that was administeredat a high dose (10 mg/kg administered orally twice a day (b.i.d.)). Thehistogram also shows that orally administered steroid (0.3 mg/kgadministered orally twice a day) increased the number of cells in BAL,and thus was not efficacious in the model.

FIGS. 4A and 4B are histograms showing the differential cell counts formacrophages (4A) or neutrophils (4B) in BAL for certain study groupsthat are shown in FIGS. 1 and 2. FIG. 4A shows that local administrationof anti-TNFR1 dAb monomer (DOM/ADS101-native (Dom1 in FIG. 1)) to thelung by intranasal administration (1 mg/kg administered once each day(q.d.)), and systemic administration of PEGylated anti-TNFR1 dAb monomer(DOM/ADS101-pegylated (TNFR1 in FIG. 2)) by intraperitonealadministration (10 mg/kg administered once every two days (q.a.d.)) weremore efficacious in reducing the number of macrophages in BAL thanphosphodiesterase 4 inhibitor (PDE4I) that was administered at a highdose (10 mg/kg administered orally twice a day (b.i.d.)). Similarly,FIG. 4B shows that local administration of anti-TNFR1 dAb monomer(DOM/ADS1010-native (Dom1 in FIG. 1)) to the lung by intranasaladministration (1 mg/kg administered once each day (q.d.)), and systemicadministration of PEGylated anti-TNFR1 dAb monomer (DOM/ADS101-pegylated(TNFR1 in FIG. 2)) by intraperitoneal administration (10 mg/kgadministered once every two days (q.a.d.)) were more efficacious inreducing the number of neutrophils in BAL than phosphodiesterase 4inhibitor (PDE4I) that was administered at a high dose (10 mg/kgadministered orally twice a day (b.i.d.)).

FIG. 5 is a graph showing the results of the pharmacokinetic study of anagent that binds TNFR1 (DOM1m (TAR2m21-23)) following localadministration to pulmonary tissue by intranasal administration (see,Example 3). The graph shows that the levels of DOM1m in lung tissueremained relatively constant for at least 8 hours after administration,while the levels in BAL declined gradually, and the levels in serumrapidly declined and were undetectable after 5 hours. Maximum levels ofDOM1m in BAL and serum were detected 1 hour after administration. (about14 μg/ml in BAL, about 150 ng/ml in serum). The levels in the BALremained high for a prolonged period of time, and gradually declinedover 24 hours (>10-fold decline after 24 hours). The levels in serumrapidly declined, and DOM1m was not detectable in serum after 5 hours.The levels of DOM1 in lung tissue were relatively constant for at least8 hours after administration, and were undetectable 24 hours afteradministration.

FIG. 6A-6V shows the amino acid sequences (SEQ ID NOS:1-198) of severalhuman immunoglobulin variable domains that have binding specificity forhuman TNFR1. The presented amino acid sequences are continuous with nogaps; the symbol ˜ has been inserted into the sequences to indicate thelocations of the complementarity determining regions (CDRs). CDR1 isflanked by ˜, CDR2 is flanked by ˜˜, and CDR3 is flanked by ˜˜˜.

FIG. 7A-7B shows the amino acid sequences (SEQ ID NOS:199-211) ofseveral human immunoglobulin variable domains that have bindingspecificity for mouse TNFR1. The presented amino acid sequences arecontinuous with no gaps; the symbol ˜ has been inserted into thesequences to indicate the locations of the complementarity determiningregions (CDRs). CDR1 is flanked by ˜˜, CDR2 is flanked by ˜˜, and CDR3is flanked by ˜˜˜.

FIG. 8A shows a nucleotide sequence (SEQ ID NO:212) encoding theextracellular domain of human (homo sapiens) TNFR1.

FIG. 8B shows the amino acid sequence (SEQ ID NO:213) of theextracellular domain of human (homo sapiens) TNFR1.

FIG. 9A shows a nucleotide sequence (SEQ ID NO:214) encoding theextracellular domain of mouse (Mus musculus) TNFR1.

FIG. 9B shows the amino acid sequence (SEQ ID NO:215) of theextracellular domain of mouse (Mus musculus) TNFR1.

FIG. 10A-10Q shows the amino acid sequences (SEQ ID NOS:216-221) ofseveral human immunoglobulin variable domains that have bindingspecificity for mouse TNFR1, and the amino acid sequence (SEQ IDNOS:222-433) of several human immunoglobulin variable domains that havebinding specificity for human TNFR1. The presented amino acid sequencesare continuous with no gaps; the symbol has been inserted into thesequences to indicate the locations of the complementarity determiningregions (CDRs). CDR1 is flanked by ˜, CDR2 is flanked by ˜˜, and CDR3 isflanked by ˜˜˜.

FIGS. 11A and 11B are graphs showing time dependent increases in theTNFα concentration in bronchoalveolar lavage (BAL) (FIG. 11A) or lungtissue (FIG. 11B) following intranasal (i.n.) administration of murineTNFα (1 mg/mouse) one hour after administration (i.n.) of vehicle oranti-TNFR1 dAb (1 mg/kg).

FIG. 12 is a graph showing time dependent increases in BAL neutrophilsfollowing i.n. administration of murine TNFα (1 μg/mouse) one hourfollowing pre-administration (i.n.) of vehicle or anti-TNFR1 dAb (1mg/kg). Pre-administration of anti-TNFR1 dAb partially inhibited theincrease in neutrophils induced by TNFα.

FIGS. 13A-13D are graphs showing time dependent effects of murine TNFαon BAL KC levels (13A), BAL MIP-2 levels (13B), BAL MCP-1 levels (13C),or lung tissue E-selectin levels (13D). Administration of anti-TNFR1 dAbsignificantly inhibited the increases induced by TNFα.

FIGS. 14A-14Z and 14A2-14J2 show the nucleotide sequences of severalnucleic acids that encode human immunoglobulin variable domains thathave binding specificity for human TNFR1, (SEQ ID NOS:434-644), and thenucleotide sequences of several nucleic acids that encode humanimmunoglobulin variable domains that have binding specificity for mouseTNFR1 (SEQ ID NOS:645-650). The presented sequences are continuous withno gaps; the symbol ˜ has been inserted into the sequences to indicatethe locations that encode the complementarity determining regions(CDRs). CDR1 is flanked by ˜, CDR2 is flanked by ˜˜, and CDR3 is flankedby ˜˜˜.

DETAILED DESCRIPTION OF THE INVENTION

As used herein, the term “antagonist” refers to an agent (e.g., amolecule, a compound) which binds a target (e.g., a receptor protein)and can inhibit a (i.e., one or more) function of the target. Forexample, an antagonist of a receptor protein can bind the receptorprotein and inhibit the binding of a natural or cognate ligand to thereceptor protein and/or inhibit signal transduction mediated throughreceptor protein. For example, antagonists of Tumor Necrosis FactorReceptor 1 “TNFR1” can bind TNFR1 and inhibit binding of TNFα to TNFR1and/or inhibit signal transduction mediated through TNFR1. Antagonistscan be identified, for example, by screening libraries or collections ofmolecules, such as, the Chemical Repository of the National CancerInstitute, or using other suitable methods. Preferred antagonists are“ligands” as described herein.

As used herein, the term “antagonist of Tumor Necrosis Factor Receptor 1(TNFR1)” refers to an agent (e.g., a molecule, a compound) which bindsTNFR1 and can inhibit a (i.e., one or more) function of TNFR1. Forexample, an antagonist of TNFR1 can inhibit the binding of TNFα to TNFR1and/or inhibit signal transduction mediated through TNFR1. Accordingly,TNFR1-mediated processes and cellular responses (e.g., TNFα-induced celldeath in a standard L929 cytotoxicity assay) can be inhibited with anantagonist of TNFR1. An antagonist of TNFR1 can be, for example, a smallorganic molecule, natural product, protein, peptide or peptidomimetic.Antagonists of TNFR1 can be identified, for example, by screeninglibraries or collections of molecules, such as, the Chemical Repositoryof the National Cancer Institute, as described herein or using othersuitable methods. Preferred antagonists of TNFR1 are antibodies,antigen-binding fragments of antibodies, ligands and dAb monomersdescribed herein.

As used herein, the term “ligand” refers to a polypeptide that comprisesa domain that has binding specificity for a desired target. Preferablythe binding domain is an immunoglobulin single variable domain (e.g.,V_(H), V_(L), V_(HH)) that has binding specificity for a desired targetantigen (e.g., a receptor protein). The binding domain can alsocomprises one or more complementarity determining regions (CDRs) of animmunoglobulin single variable domain that has binding specificity for adesired target antigen in a suitable format, such that the bindingdomain has binding specificity for the target antigen. For example, theCDRs can be grafted onto a suitable protein scaffold or skeleton, suchas an affibody, an SpA scaffold, an LDL receptor class A domain or anEGF domain. Further, the ligand can be monovalent (e.g., a dAb monomer),bivalent (homobivalent, heterobivalent) or multivalent (homomultivalent,heteromultivalent) as described herein. Thus, “ligands” includepolypeptides that consist of a dAb, include polypeptides that consistessentially of such a dAb, polypeptides that comprise a dAb (or the CDRsof a dAb) in a suitable format, such as an antibody format (e.g.,IgG-like format, scFv, Fab, Fab′, F(ab′)₂) or a suitable proteinscaffold or skeleton, such as an affibody, an SpA scaffold, an LDLreceptor class A domain or an EGF domain, dual specific ligands thatcomprise a dAb that binds a first target protein, antigen or epitope(e.g., TNFR1) and a second dAb that binds another target protein,antigen or epitope (e.g., serum albumin), and multispecific ligands asdescribed herein. The binding domain can also be a protein domaincomprising a binding site for a desired target, e.g., a protein domainis selected from an affibody, an SpA domain, an LDL receptor class Adomain an EGF domain, an avimer (see, e.g., U.S. Patent ApplicationPublication Nos. 2005/0053973, 2005/0089932, 2005/0164301).

The phrase “immunoglobulin single variable domain” refers to an antibodyvariable region (V_(H), V_(HH), V_(L)) that specifically binds anantigen or epitope independently of other V regions or domains; however,as the term is used herein, an immunoglobulin single variable domain canbe present in a format (e.g., homo- or hetero-multimer) with othervariable regions or variable domains where the other regions or domainsare not required for antigen binding by the single immunoglobulinvariable domain (i.e., where the immunoglobulin single variable domainbinds antigen independently of the additional variable domains).“Immunoglobulin single variable domain” encompasses not only an isolatedantibody single variable domain polypeptide, but also largerpolypeptides that comprise one or more monomers of an antibody singlevariable domain polypeptide sequence. A “domain antibody” or “dAb” isthe same as an “immunoglobulin single variable domain” polypeptide asthe term is used herein. An immunoglobulin single variable domainpolypeptide, as used herein refers to a mammalian immunoglobulin singlevariable domain polypeptide, preferably human, but also includes rodent(for example, as disclosed in WO 00/29004, the contents of which areincorporated herein by reference in their entirety) or camelid V_(HH)dAbs. Camelid dAbs are immunoglobulin single variable domainpolypeptides which are derived from species including camel, llama,alpaca, dromedary, and guanaco, and comprise heavy chain antibodiesnaturally devoid of light chain: V_(HH). V_(HH) molecules are about tentimes smaller than IgG molecules, and as single polypeptides, they arevery stable, resisting extreme pH and temperature conditions.

As used herein, the term “dose” refers to the quantity of agent (e.g.,antagonist of TNFR1) administered to a subject all at one time (unitdose), or in two or more administrations over a defined time interval.For example, dose can refer to the quantity of agent (e.g., antagonistof TNFR1) administered to a subject over the course of one day (24hours) (daily dose), two days, one week, two weeks, three weeks or oneor more months (e.g., by a single administration, or by two or moreadministrations). The interval between doses can be any desired amountof time.

As use herein, the term “therapeutic window” refers to the range of drug(e.g., antagonist, ligand, dAb monomer) concentrations in the plasma, orin a tissue or organ (e.g., pulmonary tissue, lung) to which a drug islocally administered, that result in a high probability of therapeuticefficacy.

“Complementary” Two immunoglobulin domains are “complementary” wherethey belong to families of structures which form cognate pairs or groupsor are derived from such families and retain this feature. For example,a V_(H) domain and a V_(L) domain of an antibody are complementary; twoV_(H) domains are not complementary, and two V_(L) domains are notcomplementary. Complementary domains may be found in other members ofthe immunoglobulin superfamily, such as the V_(α) and V_(β) (or γ and δ)domains of the T-cell receptor. Domains which are artificial, such asdomains based on protein scaffolds which do not bind epitopes unlessengineered to do so, are non-complementary. Likewise, two domains basedon (for example) an immunoglobulin domain and a fibronectin domain arenot complementary.

“Immunoglobulin” This refers to a family of polypeptides which retainthe immunoglobulin fold characteristic of antibody molecules, whichcontains two β sheets and, usually, a conserved disulphide bond. Membersof the immunoglobulin superfamily are involved in many aspects ofcellular and non-cellular interactions in vivo, including widespreadroles in the immune system (for example, antibodies, T-cell receptormolecules and the like), involvement in cell adhesion (for example theICAM molecules) and intracellular signalling (for example, receptormolecules, such as the PDGF receptor). The present invention isapplicable to all immunoglobulin superfamily molecules which possessbinding domains. Preferably, the present invention relates toantibodies.

“Domain” A domain is a folded protein structure which retains itstertiary structure independently of the rest of the protein. Generally,domains are responsible for discrete functional properties of proteins,and in many cases may be added, removed or transferred to other proteinswithout loss of function of the remainder of the protein and/or of thedomain. By single antibody variable domain is meant a folded polypeptidedomain comprising sequences characteristic of antibody variable domains.It therefore includes complete antibody variable domains and modifiedvariable domains, for example in which one or more loops have beenreplaced by sequences which are not characteristic of antibody variabledomains, or antibody variable domains which have been truncated orcomprise N- or C-terminal extensions, as well as folded fragments ofvariable domains which retain at least in part the binding activity andspecificity of the full-length domain.

“Repertoire” A collection of diverse variants, for example polypeptidevariants which differ in their primary sequence. A library used in thepresent invention will encompass a repertoire of polypeptides comprisingat least 1000 members.

“Library” The term library refers to a mixture of heterogeneouspolypeptides or nucleic acids. The library is composed of members, eachof which have a single polypeptide or nucleic acid sequence. To thisextent, library is synonymous with repertoire. Sequence differencesbetween library members are responsible for the diversity present in thelibrary. The library may take the form of a simple mixture ofpolypeptides or nucleic acids, or may be in the form of organisms orcells, for example bacteria, viruses, animal or plant cells and thelike, transformed with a library of nucleic acids. Preferably, eachindividual organism or cell contains only one or a limited number oflibrary members. Advantageously, the nucleic acids are incorporated intoexpression vectors, in order to allow expression of the polypeptidesencoded by the nucleic acids. In a preferred aspect, therefore, alibrary may take the form of a population of host organisms, eachorganism containing one or more copies of an expression vectorcontaining a single member of the library in nucleic acid form which canbe expressed to produce its corresponding polypeptide member. Thus, thepopulation of host organisms has the potential to encode a largerepertoire of genetically diverse polypeptide variants.

“Antibody” An antibody (for example IgG, IgM, IgA, IgD or IgE) orfragment (such as a Fab, F(ab′)₂, Fv, disulphide linked Fv, scFv, closedconformation multispecific antibody, disulphide-linked scFv, diabody)whether derived from any species naturally producing an antibody, orcreated by recombinant DNA technology; whether isolated from serum,B-cells, hybridomas, transfectomas, yeast or bacteria).

“Dual-specific ligand” A ligand comprising a first immunogloulin singlevariable domain and a second immunoglobulin single variable domain asherein defined, wherein the variable regions are capable of binding totwo different antigens or two epitopes on the same antigen which are notnormally bound by a monospecific immunoglobulin. For example, the twoepitopes may be on the same hapten, but are not the same epitope orsufficiently adjacent to be bound by a monospecific ligand. The dualspecific ligands according to the invention are composed of variabledomains which have different specificities, and do not contain mutuallycomplementary variable domain pairs which have the same specificity.Dual-specific ligands and suitable methods for preparing dual-specificligands are disclosed in WO 2004/058821, WO 2004/003019, and WO03/002609, the entire teachings of each of these published internationalapplications are incorporated herein by reference.

“Antigen” A molecule that is bound by a ligand according to the presentinvention. Typically, antigens are bound by antibody ligands and arecapable of raising an antibody response in vivo. It may be apolypeptide, protein, nucleic acid or other molecule. Generally, thedual specific ligands according to the invention are selected for targetspecificity against a particular antigen. In the case of conventionalantibodies and fragments thereof, the antibody binding site defined bythe variable loops (L1, L2, L3 and H1, H2, H3) is capable of binding tothe antigen.

“Epitope” A unit of structure conventionally bound by an immunoglobulinV_(H)/V_(L) pair. Epitopes define the minimum binding site for anantibody, and thus represent the target of specificity of an antibody.In the case of a single domain antibody, an epitope represents the unitof structure bound by a variable domain in isolation.

“Universal framework” A single antibody framework sequence correspondingto the regions of an antibody conserved in sequence as defined by Kabat(“Sequences of Proteins of Immunological Interest”, US Department ofHealth and Human Services) or corresponding to the human germlineimmunoglobulin repertoire or structure as defined by Chothia and Lesk,(1987) J. Mol. Biol. 196:910-917. The invention provides for the use ofa single framework, or a set of such frameworks, which has been formedto permit the derivation of virtually any binding specificity thoughvariation in the hypervariable regions alone.

“Half-life” The time taken for the serum concentration of the ligand toreduce by 50%, in vivo, for example due to degradation of the ligandand/or clearance or sequestration of the ligand by natural mechanisms.The ligands of the invention are stabilised in vivo and their half-lifeincreased by binding to molecules which resist degradation and/orclearance or sequestration. Typically, such molecules are naturallyoccurring proteins which themselves have a long half-life in vivo. Thehalf-life of a ligand is increased if its functional activity persists,in vivo, for a longer period than a similar ligand which is not specificfor the half-life increasing molecule. Thus, a ligand specific for HSAand a target molecule is compared with the same ligand wherein thespecificity for HSA is not present, that it does not bind HSA but bindsanother molecule. For example, it may bind a second epitope on thetarget molecule. Typically, the half life is increased by 10%, 20%, 30%,40%, 50% or more. Increases in the range of 2×, 3×, 4×, 5×, 10×, 20,30×, 40×, 50× or more of the half life are possible. Alternatively, orin addition, increases in the range of up to 30×, 40, 50×, 60, 70×, 80,90×, 100×, 150× of the half life are possible.

“Substantially identical (or “substantially homologous”)” A first aminoacid or nucleotide sequence that contains a sufficient number ofidentical or equivalent (e.g., with a similar side chain, e.g.,conserved amino acid substitutions) amino acid residues or nucleotidesto a second amino acid or nucleotide sequence such that the first andsecond amino acid or nucleotide sequences have similar activities. Inthe case of antibodies, the second antibody has the same bindingspecificity and has at least 50% of the affinity of the same.

As used herein, the terms “low stringency,” “medium stringency,” “highstringency,” or “very high stringency conditions” describe conditionsfor nucleic acid hybridization and washing. Guidance for performinghybridization reactions can be found in Current Protocols in MolecularBiology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6, which isincorporated herein by reference in its entirety. Aqueous and nonaqueousmethods are described in that reference and either can be used. Specifichybridization conditions referred to herein are as follows: (1) lowstringency hybridization conditions in 6× sodium chloride/sodium citrate(SSC) at about 45° C., followed by two washes in 0.2×SSC, 0.1% SDS atleast at 50° C. (the temperature of the washes can be increased to 55°C. for low stringency conditions); (2) medium stringency hybridizationconditions in 6×SSC at about 45° C., followed by one or more washes in0.2×SSC, 0.1% SDS at 60° C.; (3) high stringency hybridizationconditions in 6×SSC at about 45° C., followed by one or more washes in0.2×SSC, 0.1% SDS at 65° C.; and preferably (4) very high stringencyhybridization conditions are 0.5M sodium phosphate, 7% SDS at 65° C.,followed by one or more washes at 0.2×SSC, 1% SDS at 65° C. Very highstringency conditions (4) are the preferred conditions and the ones thatshould be used unless otherwise specified.

As referred to herein, the term “competes” means that the binding of afirst epitope to its cognate epitope binding domain is inhibited when asecond epitope is bound to its cognate epitope binding domain. Forexample, binding may be inhibited sterically, for example by physicalblocking of a binding domain or by alteration of the structure orenvironment of a binding domain such that its affinity or avidity for anepitope is reduced.

Sequences similar or homologous (e.g., at least about 70% sequenceidentity) to the sequences disclosed herein are also part of theinvention. In some embodiments, the sequence identity at the amino acidlevel can be about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99% or higher. At the nucleic acid level, the sequence identity canbe about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99% or higher. Alternatively, substantial identity exists when thenucleic acid segments will hybridize under selective hybridizationconditions (e.g., very high stringency hybridization conditions), to thecomplement of the strand. The nucleic acids may be present in wholecells, in a cell lysate, or in a partially purified or substantiallypure form.

Calculations of “homology” or “sequence identity” or “similarity”between two sequences (the terms are used interchangeably herein) areperformed as follows. The sequences are aligned for optimal comparisonpurposes (e.g., gaps can be introduced in one or both of a first and asecond amino acid or nucleic acid sequence for optimal alignment aidnon-homologous sequences can be disregarded for comparison purposes). Ina preferred embodiment, the length of a reference sequence aligned forcomparison purposes is at least 30%, preferably at least 40%, morepreferably at least 50%, even more preferably at least 60%, and evenmore preferably at least 70%, 80%, 90%, 100% of the length of thereference sequence. The amino acid residues or nucleotides atcorresponding amino acid positions or nucleotide positions are thencompared. When a position in the first sequence is occupied by the sameamino acid residue or nucleotide as the corresponding position in thesecond sequence, then the molecules are identical at that position (asused herein amino acid or nucleic acid “homology” is equivalent to aminoacid or nucleic acid “identity”). The percent identity between the twosequences is a function of the number of identical positions shared bythe sequences, talking into account the number of gaps, and the lengthof each gap, which need to be introduced for optimal alignment of thetwo sequences.

Amino acid and nucleotide sequence alignments and homology, similarityor identity, as defined herein are preferably prepared and determinedusing the algorithm BLAST 2 Sequences, using default parameters(Tatusova, T. A. et al., FEMS Microbiol Lett, 174:187-188 (1999)).Alternatively, advantageously, the BLAST algorithm (version 2.0) isemployed for sequence alignment, with parameters set to default values.The BLAST algorithm is described in detail at the world wide web site(“www”) of the National Center for Biotechnology Information (“.ncbi”)of the National Institutes of Health (“nih”) of the U.S. government(“.gov”), in the “/Blast/” directory, in the “blast_help.html” file. Thesearch parameters are defined as follows, and are advantageously set tothe defined default parameters.

BLAST (Basic Local Alignment Search Tool) is the heuristic searchalgorithm employed by the programs blastp, blastn, blastx, tblastn, andtblastx; these programs ascribe significance to their findings using thestatistical methods of Karlin and Altschul, 1990, Proc. Natl. Acad. Sci.USA 87(6):2264-8 (see the “blast_help.html” file, as described above)with a few enhancements. The BLAST programs were tailored for sequencesimilarity searching, for example to identify homologues to a querysequence. The programs are not generally useful for motif-stylesearching. For a discussion of basic issues in similarity searching ofsequence databases, see Altschul et al. (1994).

The five BLAST programs available at the National Center forBiotechnology Information web site perform the following tasks:

“blastp” compares an amino acid query sequence against a proteinsequence database;

“blastn” compares a nucleotide query sequence against a nucleotidesequence database;

“blastx” compares the six-frame conceptual translation products of anucleotide query sequence (both strands) against a protein sequencedatabase;

“tblastn” compares a protein query sequence against a nucleotidesequence database dynamically translated in all six reading frames (bothstrands).

“tblastx” compares the six-frame translations of a nucleotide querysequence against the six-frame translations of a nucleotide sequencedatabase.

BLAST uses the following search parameters:

HISTOGRAM Display a histogram of scores for each search; default is yes.(See parameter H in the BLAST Manual).

DESCRIPTIONS Restricts the number of short descriptions of matchingsequences reported to the number specified; default limit is 100descriptions. (See parameter V in the manual page). See also EXPECT andCUTOFF.

ALIGNMENTS Restricts database sequences to the number specified forwhich high-scoring segment pairs (HSPs) are reported; the default limitis 50. If more database sequences than this happen to satisfy thestatistical significance threshold for reporting (see EXPECT and CUTOFFbelow), only the matches ascribed the greatest statistical significanceare reported. (See parameter B in the BLAST Manual).

EXPECT The statistical significance threshold for reporting matchesagainst database sequences; the default value is 10, such that 10matches are expected to be found merely by chance, according to thestochastic model of Karlin and Altschul (1990). If the statisticalsignificance ascribed to a match is greater than the EXPECT threshold,the match will not be reported. Lower EXPECT thresholds are morestringent, leading to fewer chance matches being reported. Fractionalvalues are acceptable. (See parameter E in the BLAST Manual).

CUTOFF Cutoff score for reporting high-scoring segment pairs. Thedefault value is calculated from the EXPECT value (see above). HSPs arereported for a database sequence only if the statistical significanceascribed to them is at least as high as would be ascribed to a lone HSPhaving a score equal to the CUTOFF value. Higher CUTOFF values are morestringent, leading to fewer chance matches being reported. (Seeparameter S in the BLAST Manual). Typically, significance thresholds canbe more intuitively managed using EXPECT.

MATRIX Specify an alternate scoring matrix for BLASTP, BLASTX, TBLASTNand TBLASTX. The default matrix is BLOSUM62 (Henikoff & Henikoff, 1992,Proc. Natl. Acad. Sci. USA 89(22):10915-9). The valid alternativechoices include: PAM40, PAM120, PAM250 and IDENTITY. No alternatescoring matrices are available for BLASTN; specifying the MATRIXdirective in BLASTN requests returns an error response.

STRAND Restrict a TBLASTN search to just the top or bottom strand of thedatabase sequences; or restrict a BLASTN, BLASTX or TBLASTX search tojust reading frames on the top or bottom stand of the query sequence.

FILTER Mask off segments of the query sequence that have lowcompositional complexity, as determined by the SEG program of Wootton &Federhen (1993) Computers and Chemistry 17:149-163, or segmentsconsisting of short-periodicity internal repeats, as determined by theXNU program of Claverie & States, 1993, Computers and Chemistry17:191-201, or, for BLASTN, by the DUST program of Tatusov and Lipman(see the world wide web site of the NCBI). Filtering can eliminatestatistically significant but biologically uninteresting reports fromthe blast output (e.g., hits against common acidic-, basic- orproline-rich regions), leaving the more biologically interesting regionsof the query sequence available for specific matching against databasesequences.

Low complexity sequence found by a filter program is substituted usingthe letter “N” in nucleotide sequence (e.g., “N” repeated 13 times) andthe letter “X” in protein sequences (e.g., “X” repeated 9 times).

Filtering is only applied to the query sequence (or its translationproducts), not to database sequences. Default filtering is DUST forBLASTN, SEG for other programs.

It is not unusual for nothing at all to be masked by SEG, XNU, or both,when applied to sequences in SWISS-PROT, so filtering should not beexpected to always yield all effect. Furthermore, in some cases,sequences are masked in their entirety, indicating that the statisticalsignificance of any matches reported against the unfiltered querysequence should be suspect. NCBI-gi Causes NCBI gi identifiers to beshown in the output, in addition to the accession and/or locus name.Most preferably, sequence comparisons are conducted using the simpleBLAST search algorithm provided at the NCBI world wide web sitedescribed above, in the “/BLAST” directory.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art (e.g., in cell culture, molecular genetics, nucleic acidchemistry, hybridization techniques and biochemistry). Standardtechniques are used for molecular, genetic and biochemical methods (seegenerally, Sambrook et al, Molecular Cloning: A Laboratory Manual, 2ded. (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.and Ausubel et al., Short Protocols in Molecular Biology (1999) 4^(th)Ed, John Wiley & Sons, Inc. which are incorporated herein by reference)and chemical methods.

As described herein, a study in which an antagonist of TNFR1 consistingessentially of a dAb monomer that binds TNFR1 was administered in amouse subchronic model of tobacco smoke-induced chronic obstructivepulmonary disease (COPD) was conducted. The study results revealed thatantagonists of TNFR1 (e.g., that comprise a domain antibody (dAb) thatbinds TNFR1) are effective therapeutic agents for treating respiratorydiseases (e.g., inflammation in the lung, acute lung disease, chroniclung disease (e.g., COPD)). In fact, the antagonists tested in the studywere more efficacious than high dose phosphodiesterase 4 inhibitor orsoluble TNFR1 (ENBREL® (etanercept; Immunex Corporation)) which bindsand neutralizes TNFα. The antagonists of TNFR1 studied were efficaciousin the model when administered systemically (intraperitoneal injection)or locally to the pulmonary tissue by intra-nasal administration.

Surprisingly, the study results show that local administration of anantagonists that binds a target in pulmonary tissue (e.g., TNFR1) wasmore effective at inhibiting cellular infiltration of the lungs in themodel than was systemic administration of an extended half-lifeantagonist (PEGylated dAb monomer, PEGylated to increase thehydrodynamic size and the in vivo serum half-life of the dAb monomer),even though five times more antagonist was administered systemically. Inmolar terms 2.5 times more extended half-life antagonist (PEGylated dAbmonomer, PEGylated to increase the hydrodynamic size and the in vivoserum half-life of the dAb monomer) was administered systemicallycompared to the locally administered dAb monomer.

As described herein, a further study in which the pharmacokinetics of adAb monomer that binds TNFR1 after local administration to the pulmonarytissue by intranasal administration was conducted. The results of thatstudy revealed that, following local administration to the pulmonarytissue, the dAb monomer had a long residence time in the lung, and thatthe amount of dAb monomer in the lung was substantially constant over aneight hour period. In addition, local delivery of the dAb monomer to thelung resulted in the brief presence of only a low concentration of dAbmonomer in the serum. Specifically, a maximum level of about 150 ng/mlwas detected in the serum 1 hour after administration, and no dAbmonomer was detectable in the serum after 5 hours.

This results of the pharmacokinetic study are surprising and demonstratethat an agent that binds a target in pulmonary tissue, such as anantibody or antibody fragment that binds a target in pulmonary tissue(e.g, Fab fragment, Fab′ fragment, Fv fragment (e.g., scFv, disulfidebonded Fv), F(ab′)₂ fragment, dAb) or an antagonist of a target inpulmonary tissue (e.g., ligand, dAb monomer), can be locallyadministered to pulmonary tissue to provide a long therapeutic window(for treating, suppressing, preventing, or diagnosing respiratoryconditions) in pulmonary tissue due to the long residence time of suchagents in the pulmonary tissue. The results of the pharmacokineticstudy, and the long therapeutic window provided by local administrationto the pulmonary tissue, also explain the observed superior efficacy oflocally administered antagonist of TNFR1 in the mouse model of COPD.Additionally, the observed superior efficacy of dAb monomer whenadministered locally at a lower dose, the low concentration of dAbmonomer that enters into the serum, and the rapid clearance of dAbmonomer from the serum, indicate that agents that bind a target inpulmonary tissue (e.g., antibody fragment that binds a target inpulmonary tissue (e.g, Fab fragment, Fab′ fragment, Fv fragment (e.g.,scFv, disulfide bonded Fv), F(ab′)₂ fragment, dAb) are much less likelyto produce side effects (e.g., immunosuppression, toxicity) than othertypes of therapeutic agents.

In further studies lung inflammation was induced by the inflammatorystimulator TNFα. The results of these studies demonstrate thatantagonists of TNFR1 (anti-TNFR1 dAb that inhibit binding of TNFα to thereceptor, or that do not inhibit binding of TNFα to the receptor)significantly inhibit TNFα-induced increases of other inflammatorymediators, such as the early acting neutrophil chemoattractants KC andMIP-1, and the later acting chemokine MCP-1 and adhesion moleculeE-selectin, and inhibited cellular infiltration of the lungs.

The studies described herein demonstrate that agents that bind targetsin the pulmonary tissue (e.g., antibody fragments (e.g, Fab fragment,Fab′ fragment, Fv fragment (e.g., scFv, disulfide bonded Fv), F(ab′)₂fragment, dAb), antagonists, ligands, dAb monomers) are superiortherapeutics for treating, suppressing or preventing lung inflammationand/or respiratory disease, or for diagnostic purposes, such as imaging.The results also demonstrate that even though dAb monomers have a shortin vivo serum half-life, dAbs that bind a target in pulmonary tissue andantagonists that contain such a dAb, can be locally administered topulmonary tissue to provide a long therapeutic window in pulmonarytissue due to the long residence time of such a dAb in the pulmonarytissue. Accordingly, other agents that bind a target in pulmonary tissueand have short in vivo half-lives (e.g., antibody fragments such as Fabfragments, Fab′ fragments, Fv fragments (e.g., scFvs, disulfide bondedFv)s, F(ab′)₂ fragments) can be locally administered to pulmonary tissueto provide a long therapeutic window (e.g., for treating, suppressing,preventing, or diagnosing respiratory conditions) in pulmonary tissue.

Generally, agents that bind targets in the pulmonary tissue (e.g.,antibody fragments (e.g, Fab fragment, Fab′ fragment, Fv fragment (e.g.,scFv, disulfide bonded Fv), F(ab′)₂ fragment, dAb), antagonists,ligands, dAb monomers) can be locally administered to pulmonary tissueto provide a therapeutic window in pulmonary tissue of at least about 4hours, at least about 5 hours, at least about 6 hours, at least about 7hours, at least about 8 hours, at least about 9 hours; at least about 10hours, at least about 11 hours, or at least about 12 hours.

Local Administration of Agents that Bind Targets in Pulmonary Tissue toPulmonary Tissue.

In a first aspect, the invention relates to methods for administering anagent (e.g., antibody fragment, antagonist, ligand, dAb monomer) thatbinds a target in pulmonary tissue to a subject to produce a longtherapeutic window (e.g., for treating, suppressing, preventing, ordiagnosing respiratory conditions) in pulmonary tissue. For example, atherapeutic window in pulmonary tissue of at least about 4 hours, atleast about 5 hours, at least about 6 hours, at least about 7 hours, atleast about 8 hours, at least about 9 hours, at least about 10 hours, atleast about 11 hours, or at least about 12 hours. In accordance with thefirst aspect of the invention, the agent is administered locally topulmonary tissue of a subject (e.g., a human).

An agent that binds a target in pulmonary tissue (e.g., antibodyfragments (e.g, Fab fragment, Fab′ fragment, Fv fragment (e.g., scFv,disulfide bonded Fv), F(ab′)₂ fragment, dAb), antagonists, ligands, dAbmonomers) can be locally administered to pulmonary tissue (e.g., lung)of a subject using any suitable method. For example, an agent can belocally administered to pulmonary tissue via inhalation or intranasaladministration. For inhalation or intranasal administration, the agent(antagonist of TNFR1, ligand, dAb monomer) can be administered using anebulizer, inhaler, atomizer, aerosolizer, mister, dry powder inhaler,metered dose inhaler, metered dose sprayer, metered dose mister, metereddose atomizer, or other suitable delivery device.

In some embodiments, the method comprises administering locally to thepulmonary tissue of a subject an effective amount of an agent (e.g.,antibody fragment, antagonist, ligand, dAb monomer) that has a short invivo serum half-life and binds a target in pulmonary tissue. Inaccordance with the invention, such agents (e.g., antibody fragment,antagonist, ligand, dAb monomer) can be locally administered topulmonary tissue to produce a long therapeutic window in pulmonarytissue but will not substantially accumulate in the serum. Due to theshort in vivo half-life of such agents (e.g., antibody fragment,antagonist, ligand, dAb monomer), agents that cross the pulmonaryepithelium and enter the serum will be quickly eliminated from theserum, and thus will not accumulate to levels that could produceunwanted effects (e.g., systemic side effects). For example, suitableagents (e.g., antibody fragment, antagonist, ligand, dAb monomer) thatbind a target in pulmonary tissue for use in the first aspect of theinvention can have an, in vivo serum half-life of about one second toabout 12 hours, about 12 hours or less, about 11 hours or less, about 10hours or less, about 9 hours or less, about 8 hours or less, about 7hours or less, about 6 hours or less, about 5 hours or less, about 4hours or less, about 3 hours or less, about 2 hours or less, about 1hour or less, or about 30 minutes or less. Preferred antagonists foradministration in accordance with the first aspect of the inventioncomprise a dAb that binds a target in pulmonary tissue.

Particularly preferred agents (e.g., antagonists) for use in the firstaspect of the invention are dAb monomers or antigen-binding fragments ofantibodies that bind a target in pulmonary tissue (e.g., Fab fragments,Fab′ fragments, Fv fragments (e.g., scFvs, disulfide bonded Fvs, F(ab′)₂fragments). The in vivo serum half-life of dAb monomers is about 30minutes. (See, Examples 9 and 13 of WO 2004/081026 A2.) However, asdescribed herein, local delivery of a dAb monomer that binds a target inpulmonary tissue (e.g., TNFR1) resulted in a therapeutic window in thepulmonary tissue of at least 8 hours. Similarly, the in vivo serumhalf-life of antigen-binding fragments of antibodies, particularly Fvfragments, is also short and makes them unsuitable for many in vivotherapeutic and diagnostic applications. (Peters et al., Science286(5439):434 (1999).) However, as shown by the study results describedherein, antigen-binding fragments of antibodies that bind a target inpulmonary tissue can be locally administered to pulmonary tissue toprovide a long therapeutic window (e.g., for treating, suppressing,preventing, or diagnosing respiratory conditions) in pulmonary tissue,for example, a therapeutic window of at least 8 hours.

As described herein, locally administering an agent that binds a targetin pulmonary tissue (e.g., antibody fragment, antagonist, ligand, dAbmonomer) to pulmonary tissue produces a long therapeutic window in thepulmonary tissue (lung). In some embodiments, locally administering anagent that binds a target in pulmonary tissue (e.g., antibody fragment,antagonist, ligand, dAb monomer) to pulmonary tissue produces a longtherapeutic window in pulmonary tissue (lung) that is characterized bythe presence in the lung of at least about 1%, at least about 1.25%, atleast about 1.5%, at least about 1.75%, at least about 2%, at leastabout 2.25%, at least about 2.5%, at least about 2.75%, or at leastabout 3% of the total amount of agent that was administered 4 hours, 5hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, or 12hours after administration. In some embodiments, locally administeringan agent that binds a target in pulmonary tissue (e.g., antibodyfragment, antagonist, ligand, dAb monomer) to pulmonary tissue producesa long therapeutic window in lung that is characterized by the presencein the lung as a whole (BAL and lung tissue) of at least about 40%, atleast about 35%, at least about 30%, at least about 25%, at least about20%, at least about 15%, at least about 10%, or at least about 5% of thetotal amount of agent that was administered 4 hours, 5 hours, 6 hours, 7hours, 8 hours, 9 hours, 10 hours, 11 hours, or 12 hours afteradministration.

In other embodiments, locally administering an agent that binds a targetin pulmonary tissue produces a long therapeutic window in pulmonarytissue (lung) wherein at least 50%, at least 60%, at least 70%, at least75%, at least 80%, at least 85%, at least 90%, at least 95%, at least96%, at lest 97%, at least 98%, at least 99% or more of the lung levelof agent (e.g., level achieved following administration (e.g., the lunglevel achieved 1 hour after local administration to lung)) is maintainedfor a period of at least about 4 hours, at least about 5 hocus, at leastabout 6 hours, at least about 7 hours, at least about 8 hours, at leastabout 9 hours, at least about 10 hours, at least about 11 hours, or atleast about 12 hours.

As described herein, locally administering an agent that binds a targetin pulmonary tissue (e.g., antibody fragment, antagonist, ligand, dAbmonomer) to pulmonary tissue produces a long therapeutic window in thepulmonary tissue (lung), but the agent does not substantially enter thesystemic circulation. An agent does not substantially enter thecirculation when no more than about 2%, no more than about 1.75%, nomore than about 1.5%, no more than about 1.25%, no more that about 1%,no more than about 0.75%, no more than about 0.5%, or no more than about0.25% of the total amount of agent administered, or substantially noagent, is present in the serum 5 hours after the agent is administered.In some circumstances, an agent administered as described herein mayenter the systemic circulation but not accumulate to a significant levelbecause, for example, the agent is rapidly cleared from the systemiccirculation. Accordingly, the invention provides method for locallyadministering an agent that binds a target in pulmonary tissue topulmonary tissue, wherein no significant level of agent accumulates inthe systemic circulation. The level of an agent in the systemiccirculation is not significant when no more than about 2%, no more thanabout 1.75%, no more than about 1.5%, no more than about 1.25%, no morethat about 1%, no more than about 0.75%, no more than about 0.5%, or nomore than about 0.25% of the total amount of agent administered, orsubstantially no agent, is present in the serum 5 hours after the agentis administered.

Generally, only a “low dose effective amount of an agent” (e.g.,antibody fragment, antagonist, ligand, dAb monomer) that binds a targetin pulmonary tissue need be administered locally to the pulmonary tissueof a subject. A low dose effective amount is an amount of agent that isless than the amount of the same agent that would need to beadministered systemically (i.e., effective systemic dose) to achieve thesame effect. In certain embodiments, the low dose effective amount isabout 80% or less of the effective systemic dose, about 70% or less ofthe effective systemic dose, about 60% or less of the effective systemicdose, about 50% or less of the effective systemic dose, about 40% orless of the effective systemic dose, about 30% or less of the effectivesystemic dose, about 20% or less of the effective systemic dose, about10% or less of the effective systemic dose, or about 5% or less of theeffective systemic dose.

Suitable agents that can be locally administered to pulmonary tissue inaccordance with the first aspect of the invention include agents, suchas an antibody or antigen-binding fragments of antibodies (e.g, Fabfragment, Fab′ fragment, Fv fragment (e.g., scFv, disulfide bonded Fv),F(ab′)₂ fragment, dAb) and antagonists (e.g., ligand, dAb monomer) thatbind a target in pulmonary tissue, such as TNFR1, IL-1, IL-1R, IL-4,IL-4R, IL-5, IL-6, IL-6R, IL-8, IL-8R, IL-9, IL-9R, IL-10, IL-12 IL-12R,IL-13, IL-13Rα1, IL-13Rα2, IL-15, IL-15R, IL-16, IL-17R, IL-17, IL-18,IL-18R, IL-23 IL-23R, IL-25, CD2, CD4, CD11a, CD23, CD25, CD27, CD28,CD30, CD40, CD40L, CD56, CD138, ALK5, EGFR, FcER1, TGFb, CCL2, CCL18,CEA, CR8, CTGF, CXCL12 (SDF-1), chymase, FGF, Furin, Endothelin-1,Eotaxins (e.g., Eotaxin, Eotaxin-2, Eotaxin-3), GM-CSF, ICAM-1, ICOS,IgE, IFNa, I-309, integrins, L-selectin, MIF, MIP4, MDC, MCP-1, MMPs,neutrophil elastase, osteopontin, OX-40, PARC, PD-1, RANTES, SCF, SDF-1,siglec8, TARC, TGFb, Thrombin, Tim-1, TNF, TNFR1, TRANCE, Tryptase,VEGF, VLA-4, VCAM, α4β7, CCR2, CCR3, CCR4, CCR5, CCR7, CCR8,alphavbeta6, alphavbeta 8, Cmet, or CD8.

In an embodiment, the target is selected from a protein, in the TNFsignalling cascade. Preferably, this protein target is selected from thegroup comprising TNF alpha, TNF beta, TNFR2, TRADD, FADD, Caspase-8, TNFreceptor-associated factor (TRAF), TRAF2, receptor-interacting protein(RIP), Hsp90, Cdc37, IKK alpha, IKK beta, NEMO, inhibitor of kB (IkB),NF-kB, NF-kB essential modulator, apoptosis signal-regulated kinase-1(aSMase), neutral sphingomyelinase (nSMase), ASK1, Cathepsin-B, germinalcenter kinase (GSK), GSK-3, factor-associated death domain protein(FADD), factor associated with neutral sphingomyelinase activation(FAN), FLIP, JunD, inhibitor of NF-kB kinase (IKK), MKK3, MKK4, MKK7,IKK gamma, mitogen-activated protein kinase/Erk kinase kinase (MEKK),MEKK1, MEKK3, NIK, poly(ADP-ribose) polymerase (PARP), PKC-zeta, RelA,T2K, TRAF1, TRAF5, death effector domain (DED), death domain (DD), deathinducing signalling complex (DISC), inhibitor of apoptosis protein(IAP), c-Jun N-erminal kinase (JNK), mitogen-activated protein kinase(MAPK), phosphoinositide-3OH kinase (PI3K), protein kinase A (PKA), PKB,PKB, PLAD, PTEN, rel homology domain (RHD), really interesting new gene(RING), stress-activated protein kinase (SAPK), TNF alpha-convertingenzyme (TACE), silencer of death domain protein (SODD), andTRAF-associated NF-kB activator (TANK). With regard to these preferredtargets, reference is made to WO04046189, WO04046186 and WO04046185(incorporated herein by reference) which provide guidance on theselection of antibody single variable domains for targetingintracellular targets.

Agents that bind targets in pulmonary tissue (e.g., antibody fragments(e.g, Fab fragment, Fab′ fragment, Fv fragment (e.g., scFv, disulfidebonded Fv), F(ab′)₂ fragment, dAb), antagonists, ligands, dAb monomers)can be prepared using any suitable method, such as the methods describedherein in detail with respect to antagonists (e.g., ligands, dAbmonomers) that bind TNFR1.

In some embodiments, the invention is a method for providing a longtherapeutic window in pulmonary tissue of a subject (e.g., a human) foran agent (e.g., antibody fragment, antagonist, ligand, dAb monomer) thatbinds a target (e.g., TNFR1) in pulmonary tissue, comprising selectingan agent that has a short in vivo serum half-life (e.g., less than about12 hours) and binds a target in pulmonary tissue, and administeringlocally to pulmonary tissue of the subject an effective amount or lowdose effective amount of the agent that was selected.

In particular embodiments, the first aspect of the invention is a methodfor providing a long therapeutic window in pulmonary tissue of a subject(e.g., a human) for an agent (e.g., antibody fragment, antagonist,ligand, dAb monomer) that binds a target (e.g., TNFR1) in pulmonarytissue, comprising administering locally to pulmonary tissue of thesubject an effective amount or low dose effective amount of said agent.Preferably, no more than about 10 mg/kg/day of agent is administered.For example, about 1 mg/kg/day to about 10 mg/kg/day, e.g., about 1mg/kg/day, about 2 mg/kg/day, about 3 mg/kg/day, about 4 mg/kg/day,about 5 mg/kg/day, about 6 mg/kg/day, about 7 mg/kg/day, about 8mg/kg/day, about 9 mg/kg/day, or about 10 mg/kg/day of agent isadministered. In other preferred embodiments, such as when the agent isbeing administered locally to the pulmonary tissue (lung) of a human, nomore than about 10 mg/day are administered. For example, in suchembodiments the agent can be locally administered to pulmonary tissue ata dose of about 1 mg/day to about 10 mg/day (e.g., 10 mg/day, 9 mg/day,8 mg/day, 7 mg/day, 6 mg/day, 5 mg/day, 4 mg/day, 3 mg/day, 2 mg/day, or1 mg/day). Accordingly, the agent can be locally administered topulmonary tissue at a dose of about 1 μg/kg/day to about 200 μg/kg/day(e.g., about 10 μg/kg/day, about 20 μg/kg/day, about 30 μg/kg/day, about40 μg/kg/day, about 50 μg/kg/day, about 60 μg/kg/day, about 70μg/kg/day, about 80 μg/kg/day, about 90 μg/kg/day, about 100 μg/kg/day,about 110 μg/kg/day, about 120 μg/kg/day, about 130 μg/kg/day, about 140μg/kg/day, about 150 μg/kg/day, about 160 μg/kg/day, about 170μg/kg/day, about 180 μg/kg/day, or about 190 g/kg/day). In particularembodiments, about 5 μg/kg/day to about 3 mg/kg/day or preferably, about50 μg/kg/day to about 500 μg/kg/day are administered.

Use of Agents that Bind Targets in Pulmonary Tissue to Pulmonary Tissuefor Manufacture of Formulations and Medicaments.

The first aspect of the invention also relates to use of an agent (e.g.,antibody fragment, antagonist, ligand, dAb monomer) that binds a target(e.g., TNFR1) in pulmonary tissue, as described herein, in themanufacture of long acting or long therapeutic window formulation forlocal administration to pulmonary tissue. The long therapeutic window orlong action period in pulmonary tissue can be a period of at least about4 hours, at least about 5 hours, at least about 6 hours, at least about7 hours, at least about 8 hours, at least about 9 hours, at least about10 hours, at least about 11 hours, or at least about 12 hours.

In some embodiments, the agent (e.g., antibody fragment, antagonist,ligand, dAb monomer) used has a short in vivo serum half-life and bindsa target in pulmonary tissue. In accordance with the invention, suchagents (e.g., antibody fragment, antagonist, ligand, dAb monomer) can belocally administered to pulmonary tissue to produce a long therapeuticwindow in pulmonary tissue but will not substantially accumulate in theserum. For example, suitable agents (e.g., antibody fragment,antagonist, ligand, dAb monomer) that bind a target in pulmonary tissuefor use in the first aspect of the invention can have an in vivo serumhalf-life of about one second to about 12 hours, about 12 hours or less,about 11 hours or less, about 10 hours or less, about 9 hours or less,about 8 hours or less, about 7 hours or less, about 6 hours or less,about 5 hours or less, about 4 hours or less, about 3 hours or less,about 2 hours or less, about 1 hour or less, or about 30 minutes orless.

Particularly preferred agents (e.g., antagonists) for use in the firstaspect of the invention are dAb monomers or antigen-binding fragments ofantibodies that bind a target in pulmonary tissue (e.g., Fab fragments,Fab′ fragments, Fv fragments (e.g., scFvs, disulfide bonded Fv)s,F(ab′)₂ fragments).

In some embodiments, the invention relates to use of an agent (e.g.,antibody fragment, antagonist, ligand, dAb monomer) that binds a target(e.g., TNFR1) in pulmonary tissue, as described herein, in themanufacture of long acting or long therapeutic window formulation forlocal administration to pulmonary tissue (lung) wherein at least 50%, atleast 60%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 96%, at least 97%, at least 98%, atleast 99% or more of the lung level of agent (e.g., level achievedfollowing administration (e.g., the lung level achieved 1 hour afterlocal administration to lung)) is maintained for a period of at leastabout 4 hours, at least about 5 hours, at least about 6 hours, at leastabout 7 hours, at least about 8 hours, at least about 9 hours, at leastabout 10 hours, at least about II hours, or at least about 12 hours. Insome embodiments, the invention relates to use of an agent (e.g.,antibody fragment, antagonist, ligand, dAb monomer) that binds a target(e.g., TNFR1) in pulmonary tissue, as described herein, in themanufacture of long acting or long therapeutic window formulation forlocal administration to pulmonary tissue (lung) wherein the long actionperiod or long therapeutic window in lung that is characterized by thepresence in the lung as a whole (BAL and lung tissue) of at least about40%, at least about 35%, at least about 30%, at least about 25%, atleast about 20%, at least about 15%, at least about 10%, or at leastabout 5% of the total amount of agent that was administered 4 hours, 5hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, or 12hours after administration.

In some embodiments, the invention relates to use of an agent (e.g.,antibody fragment, antagonist, ligand, dAb monomer) that binds a target(e.g., TNFR1) in pulmonary tissue, as described herein, in themanufacture of long acting or long therapeutic window formulation forlocal administration to pulmonary tissue (lung) wherein a lung level ofat least about 1%, at least about 1.25%, at least about 1.5%, at leastabout 1.75%, at least about 2%, at least about 2.25%, at least about2.5%, at least about 2.75%, or at least about 3% of the amount of agentin the formulation (e.g., that is administered in a dose of theformulation) is present in pulmonary tissue for at least 4 hours, atleast 5 hours, at least 6 hours, at least 7 hours, at least 8 hours, atleast 9 hours, at least 10 hours, at least 11 hours, or at least 12hours (e.g., after the formulation is administered).

Preferably the agent for use in the invention does not substantiallyenter the systemic circulation (e.g., when in a long acting or longtherapeutic window formulation). As described herein, an agent does notsubstantially enter the circulation when no more than about 2%, no morethan about 1.75%, no more than about 1.5%, no more than about 1.25%, nomore that about 1%, no more than about 0.75%, no more than about 0.5%,or no more than about 0.25% of the total amount of agent administered(e.g, in a dose of the formulation), or substantially no agent, ispresent in the serum 5 hours after the agent (e.g, in a dose of theformulation) is administered. In some circumstances, an agentadministered as described herein may enter the systemic circulation butnot accumulate to a significant level because, for example, the agent israpidly cleared from the systemic circulation. Accordingly, theinvention provides for use of an agent that binds a target in pulmonarytissue to pulmonary tissue, wherein no significant level of agentaccumulates in the systemic circulation. The level of an agent in thesystemic circulation is not significant when no more than about 2%, nomore than about 1.75%, no more than about 1.5%, no more than about1.25%, no more that about 1%, no more than about 0.75%, no more thanabout 0.5%, or no more than about 0.25% of the total amount of agentadministered (e.g, in a dose of the formulation), or substantially noagent, is present in the serum 5 hours after the agent (e.g, in a doseof the formulation) is administered.

The invention also relates to use of an agent (e.g., antibody fragment,antagonist, ligand, dAb monomer) that binds a target (e.g., TNFR1) inpulmonary tissue, as described herein, in the manufacture of amedicament for local administration to pulmonary tissue of a low doseeffective amount of agent. As described herein, a low dose effectiveamount is generally about 80% or less of the effective systemic dose,about 70% or less of the effective systemic dose, about 60% or less ofthe effective systemic dose, about 50% or less of the effective systemicdose, about 40% or less of the effective systemic dose, about 30% orless of the effective systemic dose, about 20% or less of the effectivesystemic dose, about 10% or less of the effective systemic dose, orabout 5% or less of the effective systemic dose.

In some embodiments, the invention is a method for producing a longacting or long therapeutic window formulation for local administrationto pulmonary tissue comprising an agent (e.g., antibody fragment,antagonist, ligand, dAb monomer) that binds a target in pulmonarytissue, or a method for producing a medicament for local administrationto pulmonary tissue of a low dose effective amount of agent (e.g.,antibody fragment, antagonist, ligand, dAb monomer) that binds a targetin pulmonary tissue. The methods comprise (1) selecting an agent thatbinds a target in pulmonary tissue and has a short in vivo serumhalf-life (e.g., less than about 12 hours), and (2) using the selectedagent for the manufacture of a long acting or long therapeutic windowformulation for local administration to pulmonary tissue, or for themanufacture of medicament for local administration to pulmonary tissueof a low dose effective amount of agent.

The invention also relates to use of an agent that binds a target inpulmonary tissue (e.g., antibody fragment, antagonist, ligand, dAbmonomer), as described herein, for use in the manufacture of a longaction or long therapeutic window formulation for local administrationto pulmonary tissue (lung), as described herein, or in the manufactureof a medicament for local administration to pulmonary tissue of a lowdose effective amount of agent, as described herein, wherein theformulation or medicament is for administering no more agent than about10 mg/kg/day. For example, the formulation or medicament can be foradministering about 1 mg/kg/day to about 10 mg/kg/day, e.g., about 1mg/kg/day, about 2 mg/kg/day, about 3 mg/kg/day, about 4 mg/kg/day,about 5 mg/kg/day, about 6 mg/kg/day, about 7 mg/kg/day, about 8mg/kg/day, about 9 mg/kg/day, or about 10 mg/kg/day. In someembodiments, the formulation or medicament is for local administrationto the pulmonary tissue (lung) of a human, and the formulation ormedicament is for administering no more than about 10 mg/day. Forexample, the formulation or medicament can be for administering agent ata dose of about 1 mg/day to about 10 mg/day (e.g., 10 mg/day, 9 mg/day,8 mg/day, 7 mg/day, 6 mg/day, 5 mg/day, 4 mg/day, 3 mg/day, 2 mg/day, or1 mg/day). Accordingly, the formulation or medicament can be foradministering agent at a dose of about 1 μg/kg/day to about 200μg/kg/day (e.g., about 10 μg/kg/day, about 20 μg/kg/day, about 30μg/kg/day, about 40 μg/kg/day, about 50 μg/kg/day, about 60 μg/kg/day,about 70 μg/kg/day, about 80 μg/kg/day, about 90 μg/kg/day, about 100μg/kg/day, about 110 μg/kg/day, about 120 μg/kg/day, about 130μg/kg/day, about 140 μg/kg/day, about 150 g/kg/day, about 160 μg/kg/day,about 170 μg/kg/day, about 180 μg/kg/day, or about 190 μg/kg/day). Inparticular embodiments, about 5 μg/kg/day to about 3 mg/kg/day orpreferably, about 50 μg/kg/day to about 500 μg/kg/day are administered.

The formulations and medicaments produced using all agent that binds atarget in pulmonary tissue, as described herein, can be locallyadministered to pulmonary tissue (e.g., lung) of a subject using anysuitable method. For example, an agent can be locally administered topulmonary tissue via inhalation or intranasal administration. Forinhalation or intranasal administration, the agent (antagonist of TNFR1,ligand, dAb monomer) can be administered using a nebulizer, inhaler,atomizer, aerosolizer, mister, dry powder inhaler, metered dose inhaler,metered dose sprayer, metered dose mister, metered dose atomizer, orother suitable delivery device.

If desired, for example for diagnostic purposes (e.g. imaging), theagent that binds a target in pulmonary tissue (e.g., antibody fragment,antagonist, ligand, dAb monomer) can comprise a detectable label.Suitable detectable labels and methods for labeling an agent are wellknown in the art. Suitable detectable labels include, for example, aradioisotope (e.g., as Indium-111, Technetium-99 m or Iodine-131),positron emitting labels (e.g., Fluorine-19), paramagnetic ions (e.g.,Gadolinium (III), Manganese (II)), an epitope label (tag), an affinitylabel (e.g., biotin, avidin), a spin label, an enzyme, a fluorescentgroup or a chemiluminescent group. When labels are not employed, complexformation can be determined by surface plasmon resonance or othersuitable methods.

Antagonists of TNFR1 for Treating, Suppressing or Preventing LungInflammation and Respiratory Diseases.

In a second aspect, the invention relates to methods for treating,suppressing or preventing lung inflammation and/or a respiratory diseasecomprising administering to a subject (e.g., a mammal, a human) in needthereof an effective amount of an antagonist of TNFR1 (e.g., a ligand, adAb monomer). The invention also relates to the use of an antagonist ofTNFR1 (e.g., a ligand, a dAb monomer) for the manufacture of amedicament for treating, suppressing or preventing lung inflammationand/or respiratory disease, and to a pharmaceutical composition fortreating, suppressing or preventing lung inflammation and/or respiratorydisease comprising an antagonist of TNFR1 (e.g., a ligand, a dAbmonomer) as an active ingredient. Antagonists of TNFR1 suitable for usein the invention are described in detail herein and include smallmolecules, new chemical entities, ligands, dAb monomers, and the like.

The invention provides compositions comprising an antagonist of TNFR1(e.g. ligand, dual-specific ligand, multi-specific ligand, dAb monomer)and a pharmaceutically acceptable carrier, diluent or excipient, andtherapeutic and diagnostic methods that employ the ligands orcompositions of the invention. Antagonists and ligands (e.g.,dual-specific ligands, multispecific ligands, dAb monomers) according tothe method of the present invention may be employed in in vivotherapeutic and prophylactic applications, in vivo diagnosticapplications and the like.

Therapeutic and prophylactic uses of antagonists of TNFR1 (e.g.,ligands, multispecific ligands, dual-specific ligands, dAb monomers)comprise administering all effective amount of antagonists of TNFR1(e.g., ligands, multispecific ligands, dual-specific ligands, dAbmonomers) to a recipient mammal or subject, such as a human.

For example, the antagonists of TNFR1 (e.g., ligands, multispecificligands, dual-specific ligands, dAb monomers) will typically find use inpreventing, suppressing or treating lung inflammation and/or respiratorydiseases, such as a condition in which lung inflammation is a symptom orpart of the pathology, acute respiratory diseases, chronic respiratorydiseases, acute inflammatory respiratory diseases and chronicinflammatory respiratory diseases. For example, the antagonists of TNFR1(e.g., ligands, multispecific ligands, dual-specific ligands, dAbmonomers) can be administered to treat, suppress or prevent lunginflammation, chronic obstructive pulmonary disease (e.g., chronicbronchitis, chronic obstructive bronchitis, emphysema), asthma (e.g.,steroid resistant asthma), pneumonia (e.g., bacterial pneumonia, such asStaphylococcal pneumonia), hypersensitivity pneumonitis, pulmonaryinfiltrate with eosinophilia, environmental lung disease, pneumonia,bronchiectasis, cystic fibrosis, interstitial lung disease, primarypulmonary hypertension, pulmonary thromboembolism, disorders of thepleura, disorders of the mediastinum, disorders of the diaphragm,hypoventilation, hyperventilation, sleep apnea, acute respiratorydistress syndrome, mesothelioma, sarcoma, graft rejection, graft versushost disease, lung cancer, allergic rhinitis, allergy, asbestosis,aspergilloma, aspergillosis, bronchiectasis, chronic bronchitis,emphysema, eosinophilic pneumonia, idiopathic pulmonary fibrosis,invasive pneumococcal disease (IPD), influenza, nontuberculousmycobacteria, pleural effusion, pneumoconiosis, pneumocytosis,pneumonia, pulmonary actinomycosis, pulmonary alveolar proteinosis,pulmonary anthrax, pulmonary edema, pulmonary embolus, pulmonaryinflammation, pulmonary histiocytosis X (eosinophilic granuloma),pulmonary hypertension, pulmonary nocardiosis, pulmonary tuberculosis,pulmonary veno-occlusive disease, rheumatoid lung disease, sarcoidosis,Wegener's granulomatosis, and non-small cell lung carcinoma.

In the instant application, the term “prevention” involvesadministration of the protective composition prior to the induction ofthe disease. “Suppression” refers to administration of the compositionafter an inductive event, but prior to the clinical appearance of thedisease. “Treatment” involves administration of the protectivecomposition after disease symptoms become manifest.

Advantageously, dual- or multi-specific ligands may be used to targetcytokines and other molecules which cooperate synergistically intherapeutic situations in the body of an organism. The inventiontherefore provides a method for synergising the activity of two or morebinding domains (e.g., dAbs) wherein one domain binds TNFR1 or othertarget in pulmonary tissue, and the other domain binds a cytokine orother molecules, comprising administering a dual- or multi-specificligand capable of binding to said two or more molecules (e.g., TNFR1 anda cytokine). For example, this aspect of the invention relates tocombinations of V_(H) domains and V_(L) domains, V_(H) domains only andV_(L) domains only.

Synergy in a therapeutic context may be achieved in a number of ways.For example, target combinations may be therapeutically active only ifboth targets are targeted by the ligand, whereas targeting one targetalone is not therapeutically effective. In another embodiment, onetarget alone may provide some low or minimal therapeutic effect, buttogether with a second target the combination provides a synergisticincrease in therapeutic effect.

Animal model systems which can be used to screen the effectiveness ofthe antagonists of TNFR1 (e.g, ligands, antibodies or binding proteinsthereof, dAb monomer) in preventing, suppressing or treating respiratorydisease are available. For example, suitable animal models ofrespiratory disease include models of chronic obstructive pulmonarydisease (see, Groneberg, D A et al., Respiratory Research 5:18 (2004)),and models of asthma (see, Coffman et al., J. Exp. Med.201(12):1875-1879 (2001). Preferably, the antagonist of TNFR1 (e.g.,ligand or dAb monomer) is efficacious in a mouse tobacco smoke-inducedmodel of chronic obstructive pulmonary disease (e.g., the subchronicmodel disclosed herein) or a suitable primate model of asthma or chronicobstructive pulmonary disease. More preferably, the antagonist of TNFR1(e.g., ligand or dAb monomer) is efficacious in a mouse tobaccosmoke-induced model of chronic obstructive pulmonary disease (e.g., thesubchronic model disclosed herein) (See, also, Wright and Churg, Chest,122:301-306 (2002)). For example, administering an effective amount ofthe ligand can reduce, delay or prevent onset of the symptoms of COPD inthe model, as compared to a suitable control. The prior art does notsuggest using antagonists of TNFR1 (e.g., ligands or dAb monomers) inthese models, or that they would be efficacious.

Generally, the present antagonists (e.g., ligands) will be utilised inpurified form together with pharmacologically appropriate carriers.Typically, these carriers include aqueous or alcoholic/aqueoussolutions, emulsions or suspensions, any including saline and/orbuffered media. Parenteral vehicles include sodium chloride solution,Ringer's dextrose, dextrose and sodium chloride and lactated Ringer's.Suitable physiologically-acceptable adjuvants, if necessary to keep apolypeptide complex in suspension, may be chosen from thickeners such ascarboxymethylcellulose, polyvinylpyrrolidone, gelatin and alginates.

Intravenous vehicles include fluid and nutrient replenishers andelectrolyte replenishers, such as those based on Ringer's dextrose.Preservatives and other additives, such as antimicrobials, antioxidants,chelating agents and inert gases, may also be present (Mack (1982)Remington's Pharmaceutical Sciences, 16th Edition). A variety ofsuitable formulations can be used, including extended releaseformulations.

The antagonists (e.g., ligands) of the present invention may be used asseparately administered compositions or in conjunction with otheragents. These can include various drugs, such as phosphodiesteraseinhibitors (e.g., inhibitors of phosphodiesterase 4), bronchodilators(e.g., beta2-agonists, aniticholinergerics, theophylline), short-actingbeta-agonists (e.g., albuterol, salbutamol, bambuterol, fenoterol,isoetherine, isoproterenol, levalbuterol, metaproterenol, pirbuterol,terbutaline and tornlate), long-acting beta-agonists (e.g., formoteroland salmeterol), short acting anticholinergics (e.g., ipratropiumbromide and oxitropium bromide), long-acting anticholinergics (e.g.,tiotropium), theophylline (e.g. short acting formulation, long actingformulation), inhaled steroids (e.g., beclomethasone, beclometasone,budesonide, flunisolide, fluticasone propionate and triamcinolone), oralsteroids (e.g., methylprednisolone, prednisolone, prednisolon andprednisone), combined short-acting beta-agonists with anticholinergics(e.g., albuterol/salbutamol/ipratropium, and fenoterol/ipratropium),combined long-acting beta-agonists with inhaled steroids (e.g.,salmeterol/fluticasone, and formoterol/budesonide) and mucolytic agents(e.g., erdosteine, acetylcysteine, bromheksin, carbocysteine,guiafenesin and iodinated glycerol), cyclosporine, antibiotics,antivirals, methotrexate, adriamycin, cisplatinum, and immunotoxins.

Pharmaceutical compositions can include “cocktails” of various cytotoxicor other agents in conjunction with the antagonists (e.g., ligands) ofthe present invention, or even combinations of ligands according to thepresent invention having different specificities, such as ligandsselected using different target antigens or epitopes, whether or notthey are pooled prior to administration.

The antagonists of TNFR1 (e.g., ligands, dAb monomers) can beadministered and or formulated together with one or more additionaltherapeutic or active agents. When an antagonist of TNFR1 (e.g, ligand,dAb monomer) is administered with an additional therapeutic agent, theantagonist of TNFR1 can be administered before, simultaneously with orsubsequent to administration of the additional agent. Generally, theantagonist of TNFR1 (e.g., ligand, dAb monomer) and additional agent areadministered in a manner that provides an overlap of therapeutic effect.

The compositions containing the present antagonists (e.g., ligands) or acocktail thereof can be administered for prophylactic and/or therapeutictreatments. In certain therapeutic applications, an adequate amount toaccomplish at least partial inhibition, suppression, modulation,killing, or some other measurable parameter, of a population of selectedcells is defined as a “therapeutically-effective dose”. For example, fortreating lung inflammation and/or a respiratory disease, asputum-inhibiting amount, a bronchial biopsy inflammation-inhibitingamount, a dyspnoea-inhibiting amount, a forced expiratory volume in onesecond (FEV (1)) increasing amount, an improvement in health statusincreasing amount, as quantified in a suitable questionnaire such as theSt. George's Respiratory Questionnaire (e.g., an improvement score of 4points).

Amounts needed to achieve these effects will depend upon the severity ofthe disease and the general state of the patient's own immune system,but generally range from 0.005 to 10.0 mg of agent, antagonist (e.g.,ligand, dAb monomer) or binding protein thereof per kilogram of bodyweight, with doses of 0.05 to 2.0 mg/kg/dose being more commonly used.In particular embodiments, the amount administered will be about 5μg/kg/dose to about 3 mg/kg/dose or preferably, about 50 μg/kg/dose toabout 500 μg/kg/dose.

For prophylactic applications, compositions containing the presentligands or cocktails thereof may also be administered in similar orslightly lower dosages, to prevent, inhibit or delay onset of disease(e.g., to sustain remission or quiescence, or to prevent acute phase).The skilled clinician will be able to determine the appropriate dosinginterval to treat, suppress or prevent disease. When an antagonist ofTNFR1 (e.g., ligand) is administered to treat, suppress or prevent lunginflammation or a respiratory disease, it can be administered up to fourtimes per day, twice weekly, once weekly, once every two weeks, once amonth, or once every two months, at a dose of, for example, about 10μg/kg to about 80 mg/kg, about 100 μg/kg to about 80 mg/kg, about 1mg/kg to about 80 mg/kg, about 1 mg/kg to about 70 mg/kg, about 1 mg/kgto about 60 mg/kg, about 1 mg/kg to about 50 mg/kg, about 1 mg/kg toabout 40 mg/kg, about 1 mg/kg to about 30 mg/kg, about 1 mg/kg to about20 mg/kg, about 1 mg/kg to about 10 mg/kg, about 10 μg/kg to about 10mg/kg, about 10 μg/kg to about 5 mg/kg, about 10 μg/kg to about 2.5mg/kg, about 1 mg/kg, about 2 mg/kg, about 3 mg/kg, about 4 mg/kg, about5 mg/kg, about 6 mg/kg, about 7 mg/kg, about 8 mg/kg, about 9 mg/kg orabout 10 μg/kg. In particular embodiments, the antagonist of TNFR1(e.g., ligand) is administered to treat, suppress or prevent lunginflammation or a respiratory disease each day, every two days, once aweek, once every two weeks or once a month at a dose of about 10 μg/kgto about 10 mg/kg (e.g., about 10 μg/kg, about 100 μg/kg, about 1 mg/kg,about 2 mg/kg, about 3 mg/kg, about 4 mg/kg, about 5 mg/kg, about 6mg/kg, about 7 mg/kg, about 8 mg/kg, about 9 mg/kg or about 10 mg/kg).In particular embodiments, about 5 μg/kg to about 3 mg/kg or preferably,about 50 μg/kg to about 500 μg/kg are administered.

The antagonist of TNFR1 (e.g, ligand) can also be administered to treat,suppress or prevent lung inflammation or a respiratory disease at adaily dose or unit dose of about 10 mg, about 9 mg, about 8 mg, about 7mg, about 6 mg, about 5 mg, about 4 mg, about 3 mg, about 2 mg or about1 mg.

Treatment or therapy performed using the compositions described hereinis considered “effective” if one or more symptoms are reduced (e.g., byat least 11% or at least one point on a clinical assessment scale),relative to such symptoms present before treatment, or relative to suchsymptoms in an individual (human or model animal) not treated with suchcomposition or other suitable control. Symptoms will vary depending uponthe disease or disorder targeted, but can be measured by an ordinarilyskilled clinician or technician. Such symptoms can be measured, forexample, by monitoring one or more physical indicators of the disease ordisorder (e.g., cellular infiltrate in lung tissue, production ofsputum, cellular infiltrate in sputum, dyspnoea, exercise tolerance,spirometry (e.g., forced vital capacity (FVC), force expiratory volumein one second (FEV (1), FEV (1)/FVC), rate or severity of diseaseexacerbation, or by an accepted clinical assessment scale, for example,the St. George's Respiratory Questionnaire. Suitable clinical assessmentscales include, for example, the severity of air flow obstructionaccording to FEV (1) (Clinical Guideline 12, Chronic ObstructivePulmonary Disease, Management of Chronic Obstructive Pulmonary Diseasein Adults in Primary and Secondary Care, National Institute for ClinicalExcellence, London (2004)), Peak Expiratory Flow (PEF) (BritishGuideline on the Management of Asthma, British Thoracic Society,Scottish Intercollegiate Guidelines Network, Revised Edition (2004)),COPD stage according to the American Thoracic Society (ATS) standard(Am. J. Respir. Crit. Care Med., 152:S77-S120 (1995), asthma impairmentclass according to the ATS standard (Am. Rev. Respir. Dis.,147:1056-1061 (1993), or other accepted clinical assessment scale asknown in the field. A sustained (e.g., one day or more, preferablylonger) reduction in disease or disorder symptoms by at least 10% or byone or more points on a given clinical scale is indicative of“effective” treatment. Similarly, prophylaxis performed using acomposition as described herein is “effective” if the onset or severityof one or more symptoms is delayed, reduced or abolished relative tosuch symptoms in a similar individual (human or animal model) nottreated with the composition.

A composition containing an antagonist (e.g., ligand) according to thepresent invention may be utilised in prophylactic and therapeuticsettings to aid in the alteration, inactivation, killing or removal of aselect target cell population in a mammal. For example, suchcompositions can be used to reduce levels of inflammatory cells in lungand/or inhibit cell infiltration of the lung.

Composition containing an antagonist (e.g., ligand) according to thepresent invention can also be used to reduce levels of inflammatorymediators such as cytokines, chemokines, cellular adhesion molecules,that are induced by inflammatory stimuli in lung. For example, dAbmonomer antagonists of TNFR1 can inhibit (i) inflammatorystimulus-induced (e.g., TNFalpha-induced) increases in the levels of theearly acting mediators, such as the neutrophil chemoattractants KC andMIP-1, and/or (ii) inhibit inflammatory stimulus-induced (e.g.,TNFalpha-induced) increases in the levels of later acting mediators,such as chemokine MCP-1 and adhesion molecule E-selectin. Othermediators such as LTB4, GRO-a, IP-10, GM-CSF, reactive oxygen species(ROS), NO and the like can be effected.

The ligands of this invention can be lyophilised for storage andreconstituted in a suitable carrier prior to use. This technique hasbeen shown to be effective with conventional immunoglobulins andart-known lyophilisation and reconstitution techniques can be employed.It will be appreciated by those skilled in the art that lyophilisationand reconstitution can lead to varying degrees of antibody activity loss(e.g. with conventional immunoglobulins, IgM antibodies tend to havegreater activity loss than IgG antibodies) and that use levels may haveto be adjusted upward to compensate. The ligands of this invention canbe lyophilised to form a dry powder for inhalation, and administered inthat form.

The route of administration of pharmaceutical compositions according tothe invention may be any of those commonly known to those of ordinaryskill in the art. The administration can be by any appropriate mode,including parenterally, intravenously, intramuscularly,intraperitoneally, transdermally, via the pulmonary route, or by directinfusion with a catheter. The dosage and frequency of administrationwill depend on the age, sex and condition of the patient, concurrentadministration of other drugs, counter indications and other parametersto be taken into account by the clinician. Administration can be local(e.g., local delivery to the lung by pulmonary administration, e.g.,intranasal administration) or systemic as indicated.

In particular embodiments, an antagonist of TNFR1 is administered viapulmonary delivery, such as by inhalation (e.g., intrabronchial,intranasal or oral inhalation, intranasal drops) or by systemic delivery(e.g., parenteral, intravenous, intramuscular, intraperitoneal,subcutaneous). In preferred embodiments, the antagonist of TNFR1 (e.g.,ligand, dAb monomer) is administered to a subject via pulmonaryadministration, such as inhalation or intranasal administration (e.g.,intrabronchial, intranasal or oral inhalation, intranasal drops). Forinhalation, the antagonist of TNFR1 (e.g., ligand, dAb monomer) can beadministered with the use of a nebulizer, inhaler, atomizer,aerosolizer, mister, dry powder inhaler, metered dose inhaler, metereddose sprayer, metered dose mister, metered dose atomizer, or othersuitable delivery device.

The invention relates to a method for treating, suppressing orpreventing lung inflammation or a respiratory disease, comprisingadministering to a subject in need thereof an effective amount of anantagonist of TNFR1, wherein said effective amount does not exceed about10 mg/kg/day, and wherein preferably the level of inflammatory cells inthe lung is reduced relative to pretreatment levels with p≦0.05, orrecruitment of inflammatory cells into the lung is inhibited relative topretreatment levels with p≦0.05. The level of inflammatory cells in thelung or recruitment of inflammatory cells into the lung can be reducedor inhibited relative to pretreatment levels by at least about 30%, atleast about 40%, at least about 50%, at least about 60%, at least about70%, at least about 80%, at least about 90%, or at least about 95%.

Preferably, statistical analysis is performed using the methodsdescribed in the Examples herein. The level of inflammatory cells in thelung or recruitment of inflammatory cells into the lung can be reducedor inhibited relative to pretreatment levels with p<0.001 in someembodiments.

Levels of cells (e.g., inflammatory cells) in the lung can be assessedusing any suitable method, such as total or differential cell counts(e.g., macrophage cell count, neutrophil cell count, eosinophil cellcount, lymphocyte cell count, epithelial cell count) in BAL, sputum orbiopsy (e.g., bronchial biopsy, lung biopsy).

The invention also relates to a method for treating a respiratorydisease comprising (1) selecting all antagonist of Tumor Necrosis FactorReceptor 1 (TNFR1) that has efficacy in a suitable animal model ofrespiratory disease when administered in an amount that does not exceedabout 10 mg/kg once per day, wherein efficacy in said animal modelexists when cellular infiltration of the lungs as assessed by total cellcount in bronchoalveolar lavage is inhibited relative to untreatedcontrol with p≦0.05; and (2) administering (e.g., locally to pulmonarytissue) an effective amount of said antagonist of TNFR1 to a subject inneed thereof.

In some embodiments, the methods described herein are employed fortreating, suppressing or preventing chronic obstructive pulmonarydisease (e.g., chronic bronchitis, chronic obstructive bronchitis,emphysema), asthma (e.g., steroid resistant asthma), pneumonia (e.g.,bacterial pneumonia, such as Staphylococcal pneumonia), or lunginflammation.

The invention also relates to the use of an antagonist of TNFR1, asdescribed herein, for the manufacture of a medicament or formulation fortreating lung inflammation or a respiratory disease described herein.The medicament can be for systemic administration and/or localadministration to pulmonary tissue.

Antagonists of TNFR1

TNFR1 is a transmembrane receptor containing an extracellular regionthat binds ligand and an intercellular domain that lacks intrinsicsignal transduction activity but can associate with signal transductionmolecules. The complex of TNFR1 with bound TNF contains three TNFR1chains and three TNF chains. (Banner et al., Cell, 73(3) 431-445(1993).) The TNF ligand is present as a trimer, which is bound by threeTNFR1 chains. (Id.) The three TNFR1 chains are clustered closelytogether in the receptor-ligand complex, and this clustering is aprerequisite to TNFR1-mediated signal transduction. In fact, multivalentagents that bind TNFR1, such as anti-TNFR1 antibodies, can induce TNFR1clustering and signal transduction in the absence of TNF and arecommonly used as TNFR1 agonists. (See, e.g., Belka et al., EMBO,14(6):1156-1165 (1995); Mandik-Nayak et al., J. Immunol, 167:1920-1928(2001).) Accordingly, multivalent agents that bind TNFR1, are generallynot effective antagonists of TNFR1 even if they block the binding ofTNFα to TNFR1.

The extracellular region of TNFR1 comprises a thirteen amino acidamino-terminal segment (amino acids 1-13 of SEQ ID NO:213 (human); aminoacids 1-13 of SEQ ID NO:215 (mouse)), Domain 1 (amino acids 14-53 of SEQID NO:213 (human); amino acids 14-53 of SEQ ID NO:215 (mouse)), Domain 2(amino acids 54-97 of SEQ ID NO:213 (human); amino acids 54-97 of SEQ IDNO:215 (mouse)), Domain 3 (amino acids 98-138 of SEQ ID NO:213 (human);amino acid 98-138 of SEQ ID NO:215 (mouse)), and Domain 4 (amino acids139-167 of SEQ ID NO:213 (human); amino acids 139-167 of SEQ ID NO:215(mouse)) which is followed by a membrane-proximal region (amino acids168-182 of SEQ ID NO:213 (human); amino acids 168-183 SEQ ID NO:215(mouse)). (See, Banner et al., Cell 73(3) 431-445 (1993) and Loetscheret al., Cell 61(2) 351-359 (1990).) Domains 2 and 3 make contact withbound ligand (TNFβ, TNFα). (Banner et al., Cell, 73(3) 431-445 (1993).)The extracellular region of TNFR1 also contains a region referred to asthe pre-ligand binding assembly domain or PLAD domain (amino acids 1-53of SEQ ID NO:213 (human); amino acids 1-53 of SEQ ID NO:215 (mouse))(The Government of the USA, WO 01/58953; Deng et al., Nature Medicine,doi: 10.1038/nm1304 (2005)).

TNFR1 is shed from the surface of cells in vivo through a process thatincludes proteolysis of TNFR1 in Domain 4 or in the membrane-proximalregion (amino acids 168-182 of SEQ ID NO:213; amino acids 168-183 of SEQID NO:215), to produce a soluble form of TNFR1. Soluble TNFR1 retainsthe capacity to bind TNFα, and thereby functions as an endogenousinhibitor of the activity of TNFα.

Antagonists of TNFR1 suitable for use in the invention (e.g., ligandsdescribed herein) that have binding specificity for Tumor NecrosisFactor Receptor 1 (TNFR1; p55; CD120a). Preferably the antagonists ofTNFR1 do not have binding specificity for Tumor Necrosis Factor 2(TNFR2), or do not substantially antagonize TNFR2. An antagonist ofTNFR1 does not substantially antagonize TNFR2 when the antagonist (1 nM,10 nM, 100 mM, 1 μM, 10 μM or 100 μM) results in no more than about 5%inhibition of TNFR2-mediated activity induced by TNFα (100 pg/ml) in astandard cell assay.

Antagonists of TNFR1 that are suitable for use in the invention areeffective therapeutics (are efficacious, have therapeutic efficacy) fortreating respiratory disease (e.g., acute respiratory disease, chronicrespiratory disease, acute inflamatory respiratory disease, chronicinflammatory respiratory disease). For example, antagonists of TNFR1that suitable for use in the invention are efficacious in models ofrespiratory diseases, when an effective amount is administered.Generally an effective amount is about 1 μg/kg to about 10 mg/kg orabout 1 mg/kg to about 10 mg/kg (e.g., about 1 mg/kg, about 2 mg/kg,about 3 mg/kg, about 4 mg/kg, about 5 mg/kg, about 6 mg/kg, about 7mg/kg, about 8 mg/kg, about 9 mg/kg, or about 10 mg/kg). Severalsuitable animal models of respiratory disease are known in the art, andare recognized by those skilled in the art as being predictive oftherapeutic efficacy in humans. For example, suitable animal models ofrespiratory disease include models of chronic obstructive pulmonarydisease (see, Groneberg, D A et al., Respiratory Research 5:18 (2004)),models of asthma (see, Coffman R L et al., J. Exp. Med.201(12):1875-1879 (2001); Van Scott, M R et al., J. App. Physiol.96:1433-1444 (2004)), and models of pulmonary fibrosis (e.g.,Venkatesan, N et al., Lung 287:1342-1347 (2004). Preferably, theantagonist of TNFR1 (e.g., ligand or dAb monomer) is efficacious in amouse tobacco-induced smoke model of chronic obstructive pulmonarydisease (e.g., the subchronic model disclosed herein) or a suitableprimate model of asthma or chronic obstructive pulmonary disease (see,e.g., Coffman R L et al., J. Exp. Med. 201(12):1875-1879 (2001); VanScott, M R et al., J. App. Physiol. 96:1433-1444 (2004)). Morepreferably, the antagonist of TNFR1 (e.g., ligand or dAb monomer) isefficacious in a mouse tobacco smoke model of chronic obstructivepulmonary disease (e.g., the subchronic model disclosed herein) (See,also, Wright and Churg, Chest, 122:301-306 (2002)). For example,administering an effective amount of the ligand can reduce, delay orprevent onset of the symptoms of COPD in the model, as compared to asuitable control. The prior art does not suggest using antagonists ofTNFR1 (e.g., ligands or dAb monomers) in these models, or that theywould be efficacious.

Suitable antagonists of TNFR1 can be monovalent or multivalent. In someembodiments, the antagonist is monovalent and contains one binding sitethat interacts with TNFR1. Monovalent antagonists bind one TNFR1 and donot induce cross-linking or clustering of TNFR1 on the surface of cellswhich can lead to activation of the receptor and signal transduction. Inparticular embodiments, the monovalent antagonist of TNFR1 competes withTAR2m-21-23 for binding to mouse TNFR1 or competes with TAR2h-205 forbinding to human TNFR1.

Multivalent antagonists of TNFR1 can contain two or more copies of aparticular binding site for TNFR1 or contain two or more differentbinding sites that bind TNFR1. For example, the antagonist of TNFR1 canbe a dimer, trimer or multimer comprising two or more copies of aparticular dAb that binds TNFR1, or two or more different dAbs that bindTNFR1. Preferably, a multivalent antagonist of TNFR1 does notsubstantially agonize TNFR1 (act as an agonist of TNFR1) in a standardcell assay (i.e., when present at a concentration of 1 μM, 10 μM, 100nM, 1 μM, 10 μM, 100 μM, 1000 μM or 5,000 μM, results in no more thanabout 5% of the TNFR1-mediated activity induced by TNFα (100 pg/ml) inthe assay).

Suitable multivalent antagonists of TNFR1 can contain two or morebinding sites for a desired epitope or domain of TNFR1. For example, amultivalent antagonist of TNFR1 can comprise two or more binding sitesthat bind the same epitope of TNFR1, or two or more binding sites thatbind different epitopes or domains of TNFR1. In one example, themultivalent antagonist of TNFR1 comprises a first binding site thatbinds a first epitope of TNFR1, and a second binding site that binds asecond different epitope of TNFR1. Preferably, such multivalentantagonists do not agonize TNFR1 when present at a concentration ofabout 1 nM, or about 10 nM, or about 100 mM, or about 1 μM, or about 10μM, in a standard L929 cytotoxicity assay or a standard HeLa IL-8 assayas described herein.

Some antagonists of TNFR1 suitable for use in the invention bind TNFR1and inhibit binding of TNFα to TNFR1. In particular embodiments, such anantagonist of TNFR1 competes with TAR2h-10-27, TAR2h-131-8, TAR2h-15-8,TAR2h-35-4, TAR2h-154-7. TAR2h-154-10 or TAR2h-185-25 for binding toTNFR1.

Some antagonist of TNFR1 suitable for use in the invention do notinhibit binding of TNFα to TNFR1, but do inhibit signal transductionmediated through TNFR1. For example, an antagonist of TNFR1 can inhibitTNFα-induced clustering of TNFR1, which precedes signal transductionthrough TNFR1. Such antagonists provide several advantages. For example,in the presence of such all antagonist, TNFα can bind TNFR1 expressed onthe surface of cells and be removed from the cellular environment, butTNFR1 mediated signal transduction will not be activated. Thus, TNFR1signal-induced production of additional TNFα and other mediators ofinflammation will be inhibited. Similarly, antagonists of TNFR1 thatbind TNFR1 and inhibit signal transduction mediated through TNFR1, butdo no inhibit binding of TNFα to TNFR1, will not inhibit theTNFα-binding and inhibiting activity of endogenously produced solubleTNFR1. Accordingly, administering such an antagonist to a mammal in needthereof can complement the endogenous regulatory pathways that inhibitthe activity TNFα and the activity of TNFR1 in vivo.

In a particular embodiment, the antagonist of TNFR1 suitable for use inthe invention (e.g., a dAb monomer or ligand) binds TNFR1 and inhibitssignal transduction mediated through TNFR1 upon binding of TNFα. Such anantagonist can inhibit signal transduction through TNFR1, but notinhibit TNFα binding to TNFR1 and/or shedding of TNFR1 to producesoluble TNFR1. Accordingly, administering such an antagonist to a mammalin need thereof can complement the endogenous regulatory pathways thatinhibit the activity TNFα and the activity of TNFR1 in vivo.

Certain antagonists of TNFR1 suitable for use in the invention (e.g.,chemical compound, new chemical entity, dAb monomer, ligand) bind TNFR1and compete with TAR2m-21-23 for binding to mouse TNFR1 or competes withTAR2h-205 for binding to human TNFR1. Other antagonists of TNFR1suitable for use in the invention (e.g., chemical compound, new chemicalentity, dAb monomer, ligand) bind TNFR1 and compete with TAR2h-131-8,TAR2h-15-8, TAR2h-35-4, TAR2h-154-7, TAR2h-154-10, TAR2h-185-25, orTAR2h-27-10 for binding to TNFR1 (e.g., human and/or mouse TNFR1).

Some antagonists (e.g., ligands, dAb monomers) are cross reactive andbind human TNFR1 and TNFR1 from another species such as an animalamenable to use in medical research. For example, a dAb monomer thatbinds human TNFR1 and mouse TNFR1. Such antagonists (e.g., ligands, dAbmonomers) provide the advantage of allowing preclinical and clinicalstudies using the same antagonist (e.g., dAb monomer) and obviate theneed to conduct preclinical studies with a suitable surrogateantagonist. Preferred examples of cross reactive antagonists bind humanTNFR1 and TNFR1 from a rodent, such as mouse, rat or guinea pig, rabbit,dog, sheep, pig, or a non-human primate such as, cynomolgus monkey orrhesus macaque.

Generally, a cross reactive antagonist of the invention binds humanTNFR1 and TNFR1 from another species with similar affinities (Iii).Preferably, the cross reactive antagonists, such as a dAb monomer, bindshuman TNFR1 and TNFR1 from another species with affinities that differby no more than about a factor of 100, a factor of 10 or a factor of 5.For example, a cross reactive dAb monomer can bind human TNFR1 with anaffinity of 1 nM and also bind to mouse, cynomolgus monkey or rhesusmacaque TNFR1 with an affinity from about 10 μM to about 100 μM, about100 μM to about 10 nM, or about 200 μM to about 5 nM.

The cross reactive antagonists, such as a dAb monomer, can bind humanTNFR1 and TNFR1 from another species (e.g., one of the non-human speciesmentioned in the preceding two paragraphs) with on rates (K_(on)) thatdiffer by no more than about a factor of 100, a factor of 10, or afactor of 5, and/or with off rates (K_(off)) that differ by no more thanabout a factor of 100, a factor 10, or a factor or 5. For example, theantagonists can be a dAb monomer that binds both human TNFR1 and TNFR1from another species with a K_(on) of about 10⁴ M/s to about 10⁵ M/sand/or a K_(off) of about 10⁻³ s⁻¹ to about 10⁻⁵ s⁻¹.

Antagonists of TNFR1 suitable for use in the invention also include, anantibody that has binding specificity for TNFR1 or an antigen-bindingfragment thereof, such as Fab fragment, Fab′ fragment, F(ab′)₂ fragmentor Fv fragment (e.g., scFV). In some embodiments, the antagonist ismonovalent, such as a dAb or a monovalent antigen-binding fragment of anantibody, such as a Fab fragment, Fab′ fragment, or Fv fragment.

Preferably, the antagonist of TNFR1 is a ligand (e.g., a dAb monomer) asdescribed herein. As described herein preferred antagonists of TNFR1suitable for use in the invention comprise a dAb that binds TNFR1 andinhibits a function of TNFR1. However, instead of comprising a “dAb,” anantagonist of TNFR1 (e.g., ligand) suitable for use in the invention cancomprise a domain that comprises the CDRs of a dAb that binds TNFR1(e.g., CDRs grafted onto a suitable protein scaffold or skeleton, eg anaffibody, all SpA scaffold, an LDL receptor class A domain or an EGFdomain) or a protein domain comprising a binding site for TNFR1, e.g.,wherein the domain is selected from an affibody, an SpA domain, an LDLreceptor class A domain, in EGF domain, an avimer. The disclosure as awhole is to be construed accordingly to provide disclosure ofantagonists, ligands and methods using such domains in place of a dAb.

Antagonists of TNFR1, including ligands according to any aspect of thepresent invention, as well as dAb monomers useful in constructing suchligands, preferably bind from their target(s) with a K_(d) of 300 nM to5 μM (ie, 3×10⁻⁷ to 5×10¹²M), preferably 50 nM to 20 pM, or 5 mM to 200pM or 1 mM to 100 pM, 1×10⁻⁷ M or less, 1×10⁻⁸ M or less, 1×10⁻⁹ M orless, 1×10⁻¹⁰ M or less, 1×10⁻¹¹ M or less; and/or a K_(off) rateconstant of 5×10⁻¹ s⁻¹ to 1×10⁻⁷ s⁻¹, preferably 1×10⁻² s⁻¹ to 1×10⁻⁶s⁻¹, or 5×10⁻³ s⁻¹ to 1×10⁻⁵ s⁻¹, or 5×10⁻¹ s⁻¹ or less, or 1×10⁻² s⁻¹or less, or 1×10⁻³ s⁻¹ or less, or 1×10⁻⁴ s⁻¹ or less, or 1×10⁵ s⁻¹ orless, or 1×10⁻⁶ s⁻¹ or less as determined by surface plasmon resonance.The K_(d) rate constant is defined as K_(off)/K_(on). Additionally oralternatively, the ligand. (e.g, dAb monomer) binds TNFR1 with amoderate or fast K_(on), and a slow K_(off). Preferably, a K_(on) ofabout 10⁴ M/s to about 10⁵ M/s, and/or a K_(off) of about 10⁻³ s⁻¹ toabout 10⁻⁵ s⁻¹.

Ligands and dAb Monomers that Bind TNFR1

Preferred antagonists of TNFR1 that are suitable for use in theinvention are ligands or dAb monomers that are efficacious in models ofrespiratory diseases when an effective amount is administered. Generallyan effective amount is about 1 mg/kg to about 10 mg/kg (e.g., about 1mg/kg, about 2 mg/kg, about 3 mg/kg, about 4 mg/kg, about 5 mg/kg, about6 mg/kg, about 7 mg/kg, about 8 mg/kg, about 9 mg/kg, or about 10mg/kg). In particular embodiments, about 5 μg/kg to about 3 mg/kg orpreferably, about 50 μg/kg to about 500 μg/kg are administered.

Several suitable animal models of respiratory disease are known in theart, and are recognized by those skilled in, the art as being predictiveof therapeutic efficacy in humans. For example, suitable animal modelsof respiratory disease include models of chronic obstructive pulmonarydisease (see, Groneberg, D A et al., Respiratory Research 5:18 (2004)),and models of asthma (see, Coffman et al, J. Exp. Med. 201(12):1875-1879(2001). Preferably, the ligand or dAb monomer is efficacious in themouse subchronic model of tobacco smoke-induced chronic obstructivepulmonary disease described herein. (See, also, Wright and Churg, Chest,122:301-306 (2002).) For example, administering an effective amount ofthe ligand can reduce, delay or prevent onset of the symptoms of COPD inthe model, as compared to a suitable control. The prior art does notsuggest using antagonists of TNFR1 (e.g., ligands or dAb monomers) inthese models, or that they would be efficacious.

Generally, suitable ligands (e.g., dAb monomer) comprise an anti-TNFR1dAb monomer (e.g., dual specific ligand comprising such a dAb) thatbinds TNFR1 with a K_(d) of 300 nM to 5 pM (ie, 3×10⁻⁷ to 5×10⁻¹²M),preferably 50 nM to 20 pM, more preferably 5 nM to 200 pM and mostpreferably 1 nM to 100 pM, for example 1×10⁻⁷ M or less, preferably1×10⁻⁸ M or less, more preferably 1×10⁻⁹ M or less, advantageously1×10⁻¹⁰ M or less and most preferably 1×10⁻¹¹ M or less; and/or aK_(off) rate constant of 5×10⁻¹s⁻¹ to 1×10⁻⁷ s⁻¹ preferably 1×10⁻² s⁻¹to 1×10⁻⁶ s⁻¹ more preferably 5×10⁻³ s⁻¹ to 1×10⁻⁵ s⁻¹, for example5×10⁻¹ s⁻¹ or less, preferably 1×10⁻² s⁻¹ or less, advantageously 1×10⁻³s⁻¹ or less, more preferably 1×10⁻⁴ s⁻¹ or less, still more preferably1×10⁻⁵ s⁻¹ or less, and most preferably 1×10⁻⁶ s⁻¹ or less as determinedby surface plasmon resonance. (The K_(d)=K_(off)/K_(on)). Certainligands or dAb monomers suitable for use in the invention specificallybind human TNFR1 with a K_(d) of 50 nM to 20 pM, and a K_(off) rateconstant of 5×10⁻¹ s⁻¹ to 1×10⁻⁷ s⁻¹, as determined by surface plasmonresonance.

Some ligands or dAb monomers inhibit binding of TNFα to TNFR1. Forexample, some ligands or dAb monomers inhibit binding of TNFα to TNFR1with an inhibitory concentration 50 (IC50) of 500 nM to 50 pM,preferably 100 nM to 50 pM, more preferably 10 nM to 100 pM,advantageously 1 nM to 100 pM; for example 50 nM or less, preferably 5nM or less, more preferably 500 pM or less, advantageously 200 pM orless, and most preferably 100 pM or less. Preferably, the TNFR1 is humanTNFR1.

Other ligands and dAb monomers do not inhibit binding of TNFα to TNFR1,but are antagonists because they inhibit signal transduction mediatedthrough TNFR1. For example, a ligand or dAb monomer can inhibitTNFα-induced clustering of TNFR1, which precedes signal transductionthrough TNFR1. For example, in certain embodiments, a ligand or dAbmonomer can bind TNFR1 and inhibit TNFR1-mediated signaling, but doesnot substantially inhibit binding of TNFα to TNFR1. In some embodiments,the ligand or dAb monomer inhibits TNFα-induced crosslinking orclustering of TNFR1 on the surface of a cell, Such ligands or dAbs(e.g., TAR2h-21-23 described herein) are advantageous because they canantagonize cell surface TNFR1 but do not substantially reduce theinhibitory activity of endogenous soluble TNFR1. For example, the ligandor dAb can bind TNFR1, but inhibit binding of TNFα to TNFR1 in areceptor binding assay by no more that about 10%, no more that about 5%,no more than about 4%, no more than about 3%, no more than about 2%, orno more than about 1%. Also, in these embodiments, the ligand or dAbinhibits TNFα-induced crosslinking of TNFR1 and/or TNFR1-mediatedsignaling in a standard cell assay by at least about 100, at least about20%, at least about 30%, at least about 40%, at least about 50%, atleast about 60%, at least about 70%, at least about 80%, at least about90%, at least about 95%, or at least about 99%. Such ligands or dAbmonomers provide several advantages, as discussed herein with respect toantagonists that have these properties. Accordingly, administering suchligand or dAb monomer to a mammal in need thereof can complement theendogenous regulatory pathways that inhibit the activity TNFα and theactivity of TNFR1 in vivo.

The ligand can be monovalent (e.g., a dAb monomer) or multivalent (e.g.,dual specific, multi-specific) as described herein. In particularembodiments, the ligand is a dAb monomer that binds TNFR1. Domainantibody monomers that bind TNFR1 have a small footprint, relative toother binding formats, such as a monoclonal antibody, for example. Thus,such a dAb monomer can selectively block a function of TNFR1, but notinterfere with other functions of TNFR1. For example, a dAb monomer thatbinds TNFR1 can antagonize TNFR1 (e.g., inhibit TNFR1 mediated signaltransduction) but not inhibit binding of TNFα to TNFR1 or shedding ofTNFR1.

In more particular embodiments, the ligand is a dAb monomer that bindsTNFR1 and competes with TAR2m-21-23 for binding to mouse TNFR1 orcompetes with TAR2h-205 for binding to human TNFR1.

In other embodiments, the ligand is multivalent and comprises two ormore dAb monomers that bind TNFR1. Multivalent ligands can contain twoor more copies of a particular dAb that binds TNFR1 or contain two ormore dAbs that bind TNFR1. For example, the ligand can be a dimer,trimer or multimer comprising two or more copies of a particular dAbthat binds TNFR1, or two or more different dAbs that bind TNFR1. In someexamples, the ligand is a homo dimer or homo trimer that comprises twoor three copies of a particular dAb that binds TNFR1, respectively.Preferably, a multivalent ligand does not substantially agonize TNFR1(act as an agonist of TNFR1) in a standard cell assay (i.e., whenpresent at a concentration of 1 nM, 10 μM, 100 μM, 10×, 10 μM, 100 μM,1000 μM or 5,000 μM, results in no more than about 5% of theTNFR1-mediated activity induced by TNFα (100 pg/ml) in the assay).

In certain embodiments, the multivalent ligand contains two or more dAbsthat bind desired epitopes or domains of TNFR1, or two or more copies ofa dAb that binds a desired epitope of TNFR1. Ligands of this type canbind TNFR1 with high avidity, and be more selective for binding to cellsthat over express TNFR1 or express TNFR1 on their surface at highdensity than other ligand formats, such as dAb monomers.

In other particular embodiments, the multivalent ligand comprises two ormore dAbs, or two or more copies of a particular dAb, that binds TNFR1.Multivalent ligands of this type can bind TNFR1 monomers with lowaffinity, but bind receptor multimers (e.g., trimers see in the receptorligand complex) with high avidity. Thus, ligands of this format can beadministered to effectively target receptors that have clustered orassociated with each other and/or ligand (e.g., TNFα) which is requiredfor TNFR1-mediated signal transduction.

Preferably, the ligand or dAb monomer neutralizes (inhibits the activityof) TNFR1 in a standard assay (e.g., the standard L929 or standard HeLaIL-8 assays described herein) with a neutralizing dose 50 (ND50) of 500nM to 50 pM, preferably 100 nM to 50 pM, more preferably 10 nM to 100pM, advantageously 1 nM to 100 pM; for example 50 nM or less, preferably5 nM or less, more preferably 500 pM or less, advantageously 200 pM orless, and most preferably 100 pM or less. In other embodiments, theligand or dAb monomer binds TNFR1 and antagonizes the activity of theTNFR1 in a standard cell assay (e.g., the standard L929 or standard HeLaIL-8 assays described herein) with an ND₅₀ of ≦100 nM, and at aconcentration of ≦10 μM the dAb agonizes the activity of the TNFR1 by≦5% in the assay.

In other embodiments, the ligand or dAb monomer specifically binds TNFR1with a K_(d) described herein and inhibits lethality in a standard mouseLPS/D-galactosamine-induced septic shock model (i.e., prevents lethalityor reduces lethality by at least about 10%, as compared with a suitablecontrol). Preferably, the dAb monomer inhibits lethality by at leastabout 25%, or by at least about 50%, as compared to a suitable controlin a standard mouse LPS/D-galactosamine-induced septic shock model whenadministered at about 5 mg/kg or more preferably about 1 mg/kg.

In particular embodiments, ligand or dAb monomer does not substantiallyagonize TNFR1 (act as an agonist of TNFR1) in a standard cell assay,such as assay the standard L929 or standard HeLa IL-8 assays describedherein (i.e., when present at a concentration of 1 nM, 10 nM, 100 nM, 1μM, 10 μM, 100 μM, 1000 μM or 5,000 μM, results in no more than about 5%of the TNFR1-mediated activity induced by TNFα (100 pg/ml) in theassay).

In other embodiments, the ligand comprises a domain antibody (dAb)monomer that specifically binds Tumor Necrosis Factor Receptor 1 (TNFR1,p55, CD120a) with a K_(d) of 300 μM to 5 μM, and comprises an amino acidsequence that is at least about 80%, at least about 85%, at least about90%, at least about 91%, at least about 92%, at least about 93%, atleast about 94%, at least about 95%, at least about 96%, at least about97%, at least about 98%, or at least about 99% homologous to the aminoacid sequence or a dAb selected from the group consisting of TAR2h-112(SEQ ID NO:1), TAR2h-13 (SEQ ID NO:2), TAR2h-14 (SEQ ID NO:3), TAR2h-16(SEQ ID NO:4), TAR2h-17 (SEQ ID NO:5), TAR2h-18 (SEQ ID NO:6), TAR2h-19(SEQ ID NO:7), TAR2h-20 (SEQ ID NO:8), TAR2h-21 (SEQ ID NO:9), TAR2h-22(SEQ ID NO:10), TAR2h-23 (SEQ ID NO:11), TAR2h-24 (SEQ ID NO:12),TAR2h-25 (SEQ ID NO:13), TAR2h-26 (SEQ ID NO:14), TAR2h-27 (SEQ IDNO:15), TAR2h-29 (SEQ ID NO:16), TAR2h-30 (SEQ ID NO:17), TAR2h-32 (SEQID NO:18), TAR2h-33 (SEQ ID NO:19), TAR2h-10-1 (SEQ ID NO:20),TAR2h-10-2 (SEQ ID NO:21), TAR2h-10-3 (SEQ ID NO:22), TAR2h-10-4 (SEQ IDNO:23), TAR2h-10-5 (SEQ ID NO:24), TAR2h-10-6 (SEQ ID NO:25), TAR2h-10-7(SEQ ID NO:26), TAR2h-10-8 (SEQ ID NO:27), TAR2h-10-9 (SEQ ID NO:28),TAR2h-10-10 (SEQ ID NO:29), TAR2h-10-11 (SEQ ID NO:30), TAR2h-10-12 (SEQID NO:31), TAR2h-10-13 (SEQ ID NO:32), TAR2h-10-14 (SEQ ID NO:33),TAR2h-10-15 (SEQ ID NO:34), TAR2h-10-16 (SEQ ID NO:35), TAR2h-10-17 (SEQID NO:36), TAR2h-10-18 (SEQ ID NO:37), TAR2h-10-19 (SEQ ID NO:38),TAR2h-10-20 (SEQ ID NO:39), TAR2h-10-21 (SEQ ID NO:40), TAR2h-10-22 (SEQID NO:41), TAR2h-10-27 (SEQ ID NO:42), TAR2h-10-29 (SEQ ID NO:43),TAR2h-10-31 (SEQ ID NO:44), TAR2h-10-35 (SEQ ID NO:45), TAR-2-10-36 (SEQID NO:46), TAR2h-10-37 (SEQ ID NO:47), TAR2h-10-38 (SEQ ID NO:48),TAR2h-10-45 (SEQ ID NO:49), TAR2-10-47 (SEQ ID NO:50), TAR2h-10-48 (SEQID NO:51), TAR2h-10-57 (SEQ ID NO:52), TAR2h-10-56 (SEQ ID NO:53),TAR2h-10-58 (SEQ ID NO:54), TAR2h-10-66 (SEQ ID NO:55), TAR2h-10-64 (SEQID NO:56), TAR2h-10-65 (SEQ ID NO:57), TAR2h-10-68 (SEQ ID NO:58),TAR2h-10-69 (SEQ ID NO:59), TAR2h-10-67 (SEQ ID NO:60), TAR2h-10-61 (SEQID NO:61), TAR2h-10-62 (SEQ ID NO:62), TAR2h-10-63 (SEQ ID NO:63),TAR2h-10-60 (SEQ ID NO:64), TAR2h-10-55 (SEQ ID NO:65), TAR2h-10-59 (SEQID NO:66), TAR2h-10-70 (SEQ ID NO:67), TAR2h-34 (SEQ ID NO:68), TAR2h-35(SEQ ID NO:69), TAR2h-36 (SEQ ID NO:70), TAR2h-37 (SEQ ID NO:71),TAR2h-38 (SEQ ID NO:72), TAR2h-39 (SEQ ID NO:73), TAR2h-40 (SEQ IDNO:74), TAR2h-41 (SEQ ID NO:75), TAR2h-42 (SEQ ID NO:76), TAR2h-43 (SEQID NO:77), TAR2h-44 (SEQ ID NO:78), TAR2h-45 (SEQ ID NO:79), TAR2h-47(SEQ ID NO:80), TAR2h-48 (SEQ ID NO:81), TAR2h-50 (SEQ ID NO:82),TAR2h-51 (SEQ ID NO:83), TAR2h-66 (SEQ ID NO:84), TAR2h-67 (SEQ IDNO:85), TAR2h-68 (SEQ ID NO:86), TAR2h-70 (SEQ ID NO:87), TAR2h-71 (SEQID NO:88), TAR2h-72 (SEQ ID NO:89), TAR2h-73 (SEQ ID NO:90), TAR2h-74(SEQ ED NO:91), TAR2h-75 (SEQ ID NO:92), TAR2h-76 (SEQ ID NO:93),TAR2h-77 (SEQ ID NO:94), TAR2h-78 (SEQ ID NO:95), TAR2h-79 (SEQ IDNO:96), TAR2h-15 (SEQ ID NO:97), TAR2h-131-8 (SEQ ID NO:98),TAR2h-131-24 (SEQ ID NO:99), TAR2h-15-8 (SEQ ID NO:100), TAR2h-15-8-1SEQ ID NO:101), TAR2h-15-8-2 (SEQ ID NO:102), TAR2h-185-23 (SEQ IDNO:103), TAR2h-154-10-5 (SEQ ID NO:104), TAR2h-14-2 (SEQ ID NO:105),TAR2h-151-8 (SEQ ID NO:106), TAR2h-152-7 (SEQ ID NO:107), TAR2h-35-4(SEQ ID NO:108), TAR2h-154-7 (SEQ ID NO:109), TAR2h-80 (SEQ ID NO:110),TAR2h-8 (SEQ ID NO:111), TAR2h-82 (SEQ ID NO:112), TAR2h-83 (SEQ IDNO:113), TAR2h-84 (SEQ ID NO:114), TAR2h-85 (SEQ ID NO:115), TAR2h-86(SEQ ID NO:116), TAR2h-87 (SEQ ID NO:117), TAR2h-88 (SEQ ID NO:118),TAR2h-89 (SEQ ID NO:119), TAR2h-90 (SEQ ID NO:120), TAR2h-91 (SEQ IDNO:121), TAR2h-92 (SEQ ID NO:122), TAR2h-93 (SEQ ID NO:123), TAR2h-94(SEQ ID NO:124), TAR2h-95 (SEQ ID NO:125), TAR2h-96 (SEQ ID NO:126),TAR2h-97 (SEQ ID NO:127), TAR2h-99 (SEQ ID NO:128), TAR2h-100 (SEQ IDNO:129), TAR2h-101 (SEQ ID NO:130), TAR2h-102 (SEQ ID NO:131), TAR2h-103(SEQ ID NO:132), TAR2h-104 (SEQ ID NO:133), TAR2h-105 (SEQ ID NO:134),TAR2h-106 (SEQ ID NO:135), TAR2h-107 (SEQ ID NO:136), TAR2h-108 (SEQ IDNO:137), TAR2h-109 (SEQ ID NO:138), TAR2h-110 (SEQ ID NO:139), TAR2h-111(SEQ ID NO:140), TAR2h-112 (SEQ ID NO:141), TAR-h-113 (SEQ ID NO:142),TAR2h-114 (SEQ ID NO:143), TAR2h-115 (SEQ ID NO:144), TAR2h-116 (SEQ IDNO:145), TAR2h-117 (SEQ ID NO:146), TAR2h-118 (SEQ ID NO:147), TAR2h-119(SEQ ID NO:148), TAR2h-120 (SEQ ID NO:149), TAR2h-121 (SEQ ID NO:150),TAR2h-122 (SEQ ID NO:151), TAR2h-123 (SEQ ID NO:152), TAR2h-124 (SEQ IDNO:153), TAR2h-125 (SEQ ID NO:154), TAR2h-126 (SEQ ID NO:155), TAR2h-127(SEQ ID NO:156), TAR2h-128 (SEQ ID NO:157), TAR2h-129 (SEQ ID NO:158),TAR2h-130 (SEQ ID NO:159), TAR2h-131 (SEQ ID NO:160), TAR2h-132 (SEQ IDNO:161), TAR2h-133 (SEQ ID NO:162), TAR2h-151 (SEQ ID NO:163), TAR2h-152(SEQ ID NO:164), TAR2h-153 (SEQ ID NO:165), TAR2h-154 (SEQ ID NO:166),TAR2h-159 (SEQ ID NO:167), TAR2h-165 (SEQ ID NO:168), TAR2h-166 (SEQ IDNO:169), TAR2h-168 (SEQ ID NO:170), TAR2h-171 (SEQ ID NO:171), TAR2h-172(SEQ ID NO:172), TAR2h-1173 (SEQ ID NO:173), TAR2h-174 (SEQ ID NO:174),TAR2h-176 (SEQ ID NO:175), TAR2h-178 (SEQ ID NO:176), TAR2h-201 (SEQ IDNO:177), TAR2h-202 (SEQ ID NO:178), TAR2h-203 (SEQ ID NO:179), TAR2h-204(SEQ ID NO:180), TAR2h-185-25 (SEQ ID NO:1.81), TAR2h-154-10 (SEQ IDNO:182), TAR2h-205 (SEQ ID NO:183), TAR2h-10 (SEQ ID NO:184), TAR2h-5(SEQ ID NO:185), TAR2h-5d1 (SEQ ID NO:186), TAR2h-5d2 (SEQ ID NO:187),TAR2h-5d3 (SEQ ID NO:188), TAR2h-5d4 (SEQ ID NO:189), TAR2h-5d5 (SEQ IDNO:190), TAR2h-5d6 (SEQ ID NO:191), TAR2h-5d7 (SEQ ID NO:192), TAR2h-5d8(SEQ ID NO:193), TAR2h-5d9 (SEQ ID NO:194), TAR2h-5d10 (SEQ ID NO:195),TAR2h-5d11 (SEQ ID NO:196), TAR2h-5d12 (SEQ ID NO:197), and TAR2h-5d13(SEQ ID NO:198).

In other embodiments, the ligand comprises a domain antibody (dAb)monomer that specifically binds Tumor Necrosis Factor Receptor 1 (TNFR1,p55, CD120a) with a K_(d) of 300 nM to 5 pM, and comprises an amino acidsequence that is at least about 80%, at least about 85%, at least about90%, at least about 91%, at least about 92%, at least about 93%, atleast about 94%, at least about 95%, at least about 96%, at least about97%, at least about 98%, or at least about 99% homologous to the aminoacid sequence of a dAb that has the amino acid sequence of any of SEQ IDNO:216 through SEQ ID NO:433.

In other embodiments, the ligand comprises a domain antibody (dAb)monomer that specifically binds Tumor Necrosis Factor Receptor 1 (TNFR1,p55, CD120a) with a K, of 300 nM to 5 pM, and comprises an amino acidsequence that is at least about 80%, at least about 85%, at least about90%, at least about 91%, at least about 92%, at least about 93%, atleast about 94%, at least about 95%, at least about 96%, at least about97%, at least about 98%, or at least about 99% homologous to the aminoacid sequence or a dAb selected from the group consisting of TAR2m-14(SEQ ID NO:199), TAR2m-15 (SEQ ID NO:200), TAR2m-19 (SEQ ID NO:201),TAR2m-20 (SEQ ID NO:202), TAR2 nm-21 (SEQ ID NO:203), TAR2m-24 (SEQ IDNO:204), TAR2m-21-23 (SEQ ID NO:205), TAR2m-21-07 (SEQ ID NO:206),TAR2m-21-43 (SEQ ID NO:207), TAR2m-21-48 (SEQ ID NO:208), TAR2m-21-10(SEQ ID NO:209), TAR2m-21-06 (SEQ ID NO:210), and TAR2m-21-17 (SEQ IDNO:211).

In some embodiments, the ligand comprises a dAb monomer that binds TNFR1and competes with any of the dAbs disclosed herein for binding to TNFR1(e.g., mouse and/or human TNFR1).

The ligand of the invention can comprises a non-immunoglobulin bindingmoiety that has binding specificity for TNFR1 and preferably inhibits afunction of TNFR1 (e.g., binding TNFα, signaling upon binding TNFα),wherein the non-immunoglobulin binding moiety comprises one, two orthree of the CDRs of a V_(H), V_(L) or V_(HH) that binds TNFR1 and asuitable scaffold. In certain embodiments, the non-immunoglobulinbinding moiety comprises CDR3 but not CDR1 or CDR2 of a V_(H), V_(L) orV_(HH) that binds TNFR1 and a suitable scaffold. In other embodiments,the non-immunoglobulin binding moiety comprises CDR1 and CDR2, but notCDR3 of a V_(H), V_(L) or V_(HH) that binds TNFR1 and a suitablescaffold. In other embodiments, the non-immunoglobulin binding moietycomprises CDR1, CDR2 and CDR3 of a V_(H), V_(L) or V_(HH) that bindsTNFR1 and a suitable scaffold. Preferably, the CDR or CDRs of the ligandof these embodiments is a CDR or CDRs of an anti-TNFR1 dAb describedherein. Preferably, the non-immunoglobulin, binding moiety comprisesone, two, or three of the CDRs of one the anti-TNFR1 dAbs disclosedherein. In other embodiments, the ligand comprises only CDR3 of a V_(H),V_(L) or V_(HH) that binds TNFR1. The non-immunoglobulin domain cancomprise an amino acid sequence that one or more regions that havesequence identity to one, two or three of the CDRs of an anti-TNFR1 dAbdescribed herein. For example, the non-immunoglobulin domain can have anamino acid sequence that contains at least about 50%, at least about60%, at least about 70%, at least about 80%, or at least about 90%sequence identity with CDR1, CDR2 and/or CDR3 of an anti-TNFR1 dAbdisclosed herein. Even more preferably, the non-immunoglobulin bindingmoiety comprises one, two, or three of the CDRs of TAR2h-131-511,TAR2h-131-193 and TAR2h-131-194.

The invention also relates to a ligand comprising a protein moiety thathas a binding site with binding specificity for TNFR1, wherein saidprotein moiety comprises an amino acid sequence that is the same as theamino acid sequence of CDR3 of an anti-TNFR1 dAb disclosed herein, suchas TAR2h-131-511, TAR2h-131-193 and TAR2h-131-194.

In some embodiments, the ligand comprising a protein moiety that has abinding site with binding specificity for TNFR1, wherein the proteinmoiety has an amino acid sequence that is the same as the amino acidsequence of CDR3 of an anti-TNFR1 dAb disclosed herein, and alsocomprises an amino acid sequence that is the same as the amino acidsequence of CDR1 and/or CDR2 of an anti-TNFR1 dAb disclosed herein, suchas TAR2h-131-511, TAR2h-131-193 and TAR2h-131-194.

In other embodiments, the ligand comprises a immunoglobulin singlevariable domain that binds TNFR1, wherein the immunoglobulin singlevariable domain that binds IL-4 differs from the amino acid sequence ofan anti-TNFR1 dAb disclosed herein at no more than 25 amino acidpositions and has a CDR1 sequence that has at least 50% identity to theCDR1 sequence of the anti-TNFR1 dAbs disclosed herein, such asTAR2h-131-511, TAR2h-131-193 and TAR2h-131-194.

In other embodiments, the ligand comprises an immunoglobulin singlevariable domain that binds TNFR1, wherein the amino acid sequence of theimmunoglobulin single variable domain that binds TNFR1 differs from theamino acid sequence of an anti-TNFR1 dAb disclosed herein at no morethan 25 amino acid positions and has a CDR2 sequence that has at least50% identity to the CDR2 sequence of the anti-TNFR1 dAbs disclosedherein, such as TAR2h-131-511, TAR2h-131-193 and TAR2h-131-194.

In other embodiments, the ligand comprises an immunoglobulin singlevariable domain that binds TNFR1, wherein the amino acid sequence of theimmunoglobulin single variable domain that binds TNFR1 differs from theamino acid sequence of an anti-TNFR1 dAb disclosed herein at no morethan 25 amino acid positions and has a CDR3 sequence that has at least50% identity to the CDR3 sequence of the anti-TNFR1 dAbs disclosedherein, such as TAR2h-131-511, TAR2h-131-193 and TAR2h-131-194.

In other embodiments, the ligand comprises an immunoglobulin singlevariable domain that binds TNFR1, wherein the amino acid sequence of theimmunoglobulin single variable domain that binds TNFR1 differs from theamino acid sequence of an anti-TNFR1 dAb disclosed herein at no morethan 25 amino acid positions and has a CDR1 sequence and a CDR2 sequencethat has at least 50% identity to the CDR1 or CDR2 sequences,respectively, of the anti-TNFR1 dabs disclosed herein, such asTAR2h-131-511, TAR-h-131-193 and TAR2h-131-194.

In other embodiments, the ligand comprises an immunoglobulin singlevariable domain that binds TNFR1, wherein the amino acid sequence of theimmunoglobulin single variable domain that binds TNFR1 differs from theamino acid sequence of an anti-TNFR1 dAb disclosed herein at no morethan 25 amino acid positions and has a CDR2 sequence and a CDR3 sequencethat has at least 50% identity to the CDR2 or CDR3 sequences,respectively, of the anti-TNFR1 dabs disclosed herein, such asTAR2h-131-511, TAR2h-131-193 and TAR2h-131-194.

In other embodiments, the ligand comprises an immunoglobulin singlevariable domain that binds TNFR1, wherein the amino acid sequence of theimmunoglobulin single variable domain that binds TNFR1 differs from theamino acid sequence of an anti-TNFR1 dAb disclosed herein at no morethan 25 amino acid positions and has a CDR1 sequence and a CDR3 sequencethat has at least 50% identity to the CDR1 or CDR3 sequences,respectively, of the anti-TNFR1 dabs disclosed herein, such asTAR2h-131-511, TAR2h-131-1.93 and TAR2h-131-194.

In other embodiments, the ligand comprises au immunoglobulin singlevariable domain that binds TNFR1, wherein the amino acid sequence of theimmunoglobulin single variable domain that binds TNFR1 differs from theamino acid sequence of an anti-TNFR1 dAb disclosed herein at no morethan 25 amino acid positions and has a CDR1 sequence, a CDR2 sequenceand a CDR3 sequence that has at least 50% identity to the CDR1, CDR2 orCDR3 sequences, respectively, of the anti-TNFR1 dAbs disclosed herein,such as TAR2h-131-511, TAR2h-131-193 and TAR2h-131-194.

In another embodiment, the invention is a ligand comprising animmunoglobulin single variable domain that binds TNFR1, wherein theimmunoglobulin single variable domain that binds TNFR1 has a CDR2sequence that has at least 50% identity to the CDR2 sequences of ananti-TNFR1 dAb disclosed herein, such as TAR2h-131-511, TAR2h-131-193and TAR2h-131-194.

In another embodiment, the invention is a ligand comprising animmunoglobulin single variable domain that binds TNFR1, wherein theimmunoglobulin single variable domain that binds TNFR1 has a CDR3sequence that has at least 50% identity to the CDR3 sequences of ananti-TNFR1 dAb disclosed herein, such as TAR2h-131-511, TAR2h-131-193and TAR2h-131-194.

In another embodiment, the invention is a ligand comprising animmunoglobulin single variable domain that binds TNFR1, wherein theimmunoglobulin single variable domain that binds TNFR1 has a CDR1 and aCDR2 sequence that has at least 50% identity to the CDR1 and CDR2sequences, respectively, of an anti-TNFR1 dAb disclosed herein, such asTAR2h-131-511, TAR2h-131-193 and TAR2h-131-194.

In another embodiment, the invention is a ligand comprising inimmunoglobulin single variable domain that binds TNFR1, wherein theimmunoglobulin single variable domain that binds TNFR1 has a CDR2 and aCDR3 sequence that has at least 50% identity to the CDR2 and CDR3sequences, respectively, of an anti-TNFR1 dAb disclosed herein, such asTAR2h-131-511, TAR2h-131-193 and TAR2h-131-194.

In another embodiment, the invention is a ligand comprising animmunoglobulin single variable domain that binds TNFR1, wherein theimmunoglobulin single variable domain that binds TNFR1 has a CDR1 and aCDR3 sequence that has at least 50% identity to the CDR1 and CDR3sequences, respectively, of an anti-TNFR1 dAb disclosed herein, such asTAR2h-131-511, TAR2h-131-193 and TAR2h-131-194.

In another embodiment, the invention is a ligand comprising animmunoglobulin single variable domain that binds TNFR1, wherein theimmunoglobulin single variable domain that binds TNFR1 has a CDR1, CDR2,and a CDR3 sequence that has at least 50% identity to the CDR1, CDR2 andCDR3 sequences, respectively, of an anti-TNFR1 dAb disclosed herein,such as TAR2h-131-511, TAR2h-131-193 and TAR2h-131-194.

In some embodiments, the ligand comprising a dAb that binds human TNFR1and is an antagonist of human TNFR1, wherein said ligand inhibitsTNFα-induced inflammation or TNFα-induced inflammatory mediator at adose [mg/kg] that is no more than ½, ⅓, ¼, ⅕, 1/10, 1/15, 1/20, 1/25, or1/30 the dose of etanercept (ENBREL, Immunex Corporation) that isrequired to inhibit said TNFα-induced inflammation or TNFα-inducedinflammatory mediator to substantially the same of to the same degree.For example, the ligand can inhibit TNFα-induced cell influx of tissue(e.g., lung), TNFα-induced increase in the production, concentration orlevel of inflammatory mediators, such as the early acting neutrophilchemoattractants KC and MIP-1, and the later acting chemokine MCP-1 andadhesion molecule E-selectin, at a dose [mg/kg] that is no more than ½,⅓, ¼, ⅕, 1/10, 1/15, 1/20, 1/25, or 1/30 the dose of etanercept (ENBREL,Immunex Corporation) that is required to achieve substantially the sameor the same level of inhibition. Preferably, the level of inhibition isat least about 50%, at least about 60%, at least about 70%, at leastabout 80%, at least about 90%, or at least about 95%.

The dAb monomer can comprise any suitable immunoglobulin variabledomain, and preferably comprises a human variable domain or a variabledomain that comprises human framework regions. In certain embodiments,the dAb monomer comprises a universal framework, as described herein.

The universal framework can be a V_(L) framework (Vλ or Vκ), such as aframework that comprises the framework amino acid sequences encoded bythe human germline DPK1, DPK2, DPK3, DPK4, DPK5, DPK6, DPK7, DPK8, DPK9,DPK10, DPK12, DPK13, DPK15, DPK16, DPK18, DPK19, DPK20, DPK21, DPK22,DPK23, DPK24, DPK25, DPK26 or DPK 28 immunoglobulin gene segment. Ifdesired, the V_(L) framework can further comprises the framework aminoacid sequence encoded by the human germline J_(κ)1, J_(κ)2, J_(κ)3,J_(κ)4, or J_(κ)5 immunoglobin gene segment.

In other embodiments the universal framework can be a V_(H) framework,such as a framework that comprises the framework amino acid sequencesencoded by the human germline DP4, DP7, DP8, DP9, DP10, DP31, DP33,DP38, DP45, DP46, DP47, DP49, DP50, DP51, DP53, DP54, DP65, DP66, DP67,DP68 or DP69 immunoglobulin gene segment. If desired, the V_(H)framework can further comprises the framework amino acid sequenceencoded by the human germline J_(H)1 J_(H)2, J_(H)3, J_(H)4, J_(H)4b,J_(H)5 and J_(H)6 immunoglobulin gene segment.

In particular embodiments, the dAb monomer comprises the DPK9 V_(L)framework, or a V_(H) framework selected from the group consisting ofDP47, DP45 and DP38.

In certain embodiments, the dAb monomer comprises one or more frameworkregions comprising an amino acid sequence that is the same as the aminoacid sequence of a corresponding framework region encoded by a humangermline antibody gene segment, or the amino acid sequences of one ormore of said framework regions collectively comprise up to 5 amino aciddifferences relative to the amino acid sequence of said correspondingframework region encoded by a human germline antibody gene segment.

In other embodiments, the amino acid sequences of FW1, FW2, FW3 and FW4of the dAb monomer are the same as the amino acid sequences ofcorresponding framework regions encoded by a human germline antibodygene segment, or the amino acid sequences of FW1, FW2, FW3 and FW4collectively contain up to 10 amino acid differences relative to theamino acid sequences of corresponding framework regions encoded by saidhuman germline antibody gene segment.

In other embodiments, the dAb monomer comprises FW1, FW2 and FW3regions, and the amino acid sequence of said FW1, FW2 and FW3 regionsare the same as the amino acid sequences of corresponding frameworkregions encoded by human germline antibody gene segments.

In some embodiments, the dAb monomer does not comprise a Camelidimmunoglobulin variable domain, or one or more framework amino acidsthat are unique to immunoglobulin) variable domains encoded by Camelidgermline antibody gene segments.

Nucleic Acid Molecules, Vectors and Host Cells

The invention also provides isolated and/or recombinant nucleic acidmolecules encoding ligands as described herein. Nucleic acids referredto herein as “isolated” are nucleic acids which have been separated awayfrom the nucleic acids of the genomic DNA or cellular RNA of theirsource of origin (e.g., as it exists in cells or in a mixture of nucleicacids such as a library), and include nucleic acids obtained by methodsdescribed herein or other suitable methods, including essentially purenucleic acids, nucleic acids produced by chemical synthesis, bycombinations of biological and chemical methods, and recombinant nucleicacids which are isolated (see e.g., Daugherty, B. L. et al., NucleicAcids Res., 19(9): 2471-2476 (1991); Lewis, A. P. and J. S. Crowe, Gene,101: 297-302 (1991)).

Nucleic acids referred to herein as “recombinant” are nucleic acidswhich have been produced by recombinant DNA methodology, including thosenucleic acids that are generated by procedures which rely upon a methodof artificial recombination, such as the polymerase chain reaction (PCR)and/or cloning into a vector using restriction enzymes.

In certain embodiments, the isolated and/or recombinant nucleic acidcomprises a nucleotide sequence encoding a ligand, as described herein,wherein said ligand comprises an amino acid sequence that has at leastabout 80%, at least about 85%, at least about 90%, at least about 91%,at least about 92%, at least about 93%, at least about 94%, at leastabout 95%, at least about 96%, at least about 97%, at least about 98%,or at least about 99% amino acid sequence identity with the amino acidsequence of a dAb that binds TNFR1 disclosed herein.

For example, in some embodiments, the isolated and/or recombinantnucleic acid comprises a nucleotide sequence encoding a ligand that hasbinding specificity for TNFR1 wherein said ligand comprises an aminoacid sequence that has at least about 80%, at least about 85%, at leastabout 90%, at least about 91%, at least about 92%, at least about 93%,at least about 94%, at least about 95%, at least about 96%, at leastabout 97%, at least about 98%, or at least about 99% amino acid sequenceidentity with the amino acid sequence of a dAb selected from the groupconsisting of TAR2m-15-8 (SEQ ID NO:216), TAR2m-15-12 (SEQ ID NO:217),TAR2 nm-15-2 (SEQ ID NO:218), TAR2m-15-5 (SEQ ID NO:219), TAR2 nm-15-6(SEQ ID NO:220), TAR2m-15-9 (SEQ ID NO:221), Tar2h-131-1 (SEQ IDNO:222), Tar2h-131-2 (SEQ ID NO:223), Tar2h-131-3 (SEQ ID NO:224),Tar2h-131-4 (SEQ ID NO:225), Tar2h-131-5 (SEQ ID NO:226), Tar2h-131-6(SEQ ID NO:227), Tar2h-131-7 (SEQ ID NO:228), Tar2h-131-8 (SEQ IDNO:229), Tar2h-131-9 (SEQ ID NO:230), Tar2h-131-10 (SEQ ID NO:231),Tar2h-131-11 (SEQ ID NO:232), Tar2h-131-12 (SEQ ID NO:233), Tar2h-131-13(SEQ ID NO:234), Tar2h-131-14 (SEQ ID NO:235), Tar2h-131-15 (SEQ IDNO:236), Tar21 h-131-16 (SEQ ID NO:237), Tar2h-131-17 (SEQ ID NO:238),Tar2h-131-18 (SEQ ID NO:239), Tar2h-131-19 (SEQ ID NO:240), Tar2h-131-20(SEQ ID NO:241), Tar2h-131-21 (SEQ ID NO:242), Tar2h-131-22 (SEQ IDNO:243), Tar2h-131-23 (SEQ ID NO:244), Tar2h-131-24 (SEQ ID NO:245),Tar2h-131-25 (SEQ ID NO:246), Tar2h-131-26 (SEQ ID NO:247), Tar2h-131-27(SEQ ID NO:248), Tar2h-131-28 (SEQ ID NO:249), Tar2h-131-29 (SEQ IDNO:250), Tar2h-131-30 (SEQ ID NO:251), Tar2h-131-31 (SEQ ID NO:252),Tar2h-131-32 (SEQ ID NO:253), Tar2h-131-33 (SEQ ID NO:254), Tar2h-131-34(SEQ ID NO:255), Tar2h-131-35 (SEQ ID NO:256), Tar2h-131-36 (SEQ IDNO:257), Tar2h-131-37 (SEQ ID NO:258), Tar2h-131-38 (SEQ ID NO:259),Tar2h-131-39 (SEQ ID NO:260), Tar2h-131-40 (SEQ ID NO:261), Tar2h-131-41(SEQ ID NO:262), Tar2h-131-42 (SEQ ID NO:263), Tar2h-131-43 (SEQ IDNO:264), Tar2h-131-44 (SEQ ID NO:265), Tar2h-131-45 (SEQ ID NO:266),Tar2h-31-46 (SEQ ID NO:267), Tar2h-131-47 (SEQ ID NO:268), Tar2h-131-48(SEQ ID NO:269), Tar2h-131-49 (SEQ ID NO:270), Tar2h-131-50 (SEQ IDNO:271), Tar2h-131-51 (SEQ ID NO:272), Tar2h-131-52 (SEQ ID NO:273),Tar2h-131-53 (SEQ ID NO:274), Tar2h-131-54 (SEQ ID NO:275), Tar2h-131-55(SEQ ID NO:276), Tar2h-1311-56 (SEQ ID NO:277), Tar2h-131-57 (SEQ IDNO:278), Tar2h-131-58 (SEQ ID NO:279), Tar2h-131-59 (SEQ ID NO:280),Tar2h-131-60 (SEQ ID NO:281), Tar2h-131-61 (SEQ ID NO:282), Tar2h-131-62(SEQ ID NO:283), Tar2h-131-63 (SEQ ID NO:284), Tar2h-131-64 (SEQ IDNO:285), Tar2h-131-65 (SEQ ID NO:286), Tar2h-131-66 (SEQ ID NO:287),Tar2h-131-67 (SEQ ID NO:288), Tar2h-131-68 (SEQ ID NO:289), Tar2h-131-69(SEQ ID NO:290), Tar2h-131-70 (SEQ ID NO:291), Tar2h-131-71 (SEQ IDNO:292), Tar2h-131-72 (SEQ ID NO:293), Tar2h-131-73 (SEQ ID NO:294),Tar21-131-74 (SEQ ID NO:295), Tar2h-131-75 (SEQ ID NO:296), Tar2h-131-76(SEQ ID NO:297), Tar2h-131-77 (SEQ ID NO:298), Tar2h-131-78 (SEQ IDNO:299), Tar2h-131-79 (SEQ ID NO:300), Tar2h-131-80 (SEQ ID NO:301),Tar2h-131-81 (SEQ ID NO:302), Tar2h-131-82 (SEQ ID NO:303), Tar2h-131-83(SEQ ID NO:304), Tar2h-131-86 (SEQ ID NO:305), Tar21 h-131-87 (SEQ IDNO:306), Tar2h-131-88 (SEQ ID NO:307), Tar2h-131-89 (SEQ ID NO:308),Tar2h-131-90 (SEQ ID NO:309), Tar2h-131-91 (SEQ ID NO:310), Tar2h-131-92(SEQ ID NO:311), Tar2h-131-93 (SEQ ID NO:312), Tar2h-131-94 (SEQ IDNO:313), Tar2h-131-95 (SEQ ID NO:314), Tar2h-131-96 (SEQ ID NO:315),Tar2h-131-97 (SEQ ID NO:316), Tar2h-131-99 (SEQ ID NO:317),Tar2h-131-100 (SEQ ID NO:318), Tar2h-131-101 (SEQ ID NO:319),Tar2h-131-102 (SEQ ID NO:320), Tar2h-131-103 (SEQ ID NO:321),Tar2h-131-104 (SEQ ID NO:322), Tar2h-131-105 (SEQ ID NO:323),Tar2h-131-106 (SEQ ID NO:324), Tar2h-131-107 (SEQ ID NO:325),Tar2h-131-108 (SEQ ID NO:326), Tar2h-131-109 (SEQ ID NO:327),Tar2h-131-110 (SEQ ID NO:328), Tar2h-131-111 (SEQ ID NO:329),Tar2h-131-112 (SEQ ID NO:330), Tar2h-131-113 (SEQ ID NO:331),Tar2h-131-114 (SEQ ID NO:332), Tar2h-131-115 (SEQ ID NO:333),Tar211-131-116 (SEQ ID NO:334), Tar2h-131-117 (SEQ ID NO:335),Tar2h-131-120 (SEQ ID NO:336), Tar2h-131-121 (SEQ ID NO:337),Tar2h-131-122 (SEQ ID NO:338), Tar2h-131-123 (SEQ ID NO:339),Tar2h-131-124 (SEQ ID NO:340), Tar2h-131-125 (SEQ ID NO:341),Tar2h-131-126 (SEQ ID NO:342), Tar2h-131-127 (SEQ ID NO:343),Tar2h-131-128 (SEQ ID NO:344), Tar2h-131-129 (SEQ ID NO:345),Tar2h-131-1.30 (SEQ ID NO:346), Tar2h-131-131 (SEQ ID NO:347),Tar2h-131-132 (SEQ ID NO:348), Tar2h-131-136 (SEQ ID NO:349),Tar2h-131-1.51 (SEQ ID NO:350), Tar2h-131-180 (SEQ ID NO:351),Tar2h-131-181 (SEQ ID NO:352), Tar2h-131-182 (SEQ ID NO:353),Tar2h-1.31-183 (SEQ ID NO:354), Tar2h-131-184 (SEQ ID NO:355),Tar2h-131-185 (SEQ ID NO:356), Tar2h-131-188 (SEQ ID NO:357),Tar2h-131-189 (SEQ ID NO:358), Tar2h-131-190 (SEQ ID NO:359),Tar2h-131-191 (SEQ ID NO:360), Tar2h-131-192 (SEQ ID NO:361),Tar2h-131-193 (SEQ ID NO:362), Tar2h-131-194 (SEQ ID NO:363),Tar2h-131-195 (SEQ ID NO:364), Tar2h-131-196 (SEQ ID NO:365),Tar2h-131-197 (SEQ ID NO:366), Tar2h-131-198 (SEQ ID NO:367),Tar2h-131-500 (SEQ ID NO:368), Tar2h-131-501 (SEQ ID NO:369),Tar2h-131-502 (SEQ ID NO:370), Tar2h-131-503 (SEQ ID NO:371),Tar2h-131-504 (SEQ ID NO:372), Tar2h-131-505 (SEQ ID NO:373),Tar2h-131-506 (SEQ ID NO:374), Tar2h-131-507 (SEQ ID NO:375),Tar2h-131-508 (SEQ ID NO:376), Tar2h-131-509 (SEQ ID NO:377),Tar2h-131-510 (SEQ ID NO:378), Tar2h-131-511 (SEQ ID NO:379),Tar2h-131-512 (SEQ ID NO:380), Tar2h-131-513 (SEQ ID NO:381),Tar2h-131-514 (SEQ ID NO:382), Tar2h-131-515 (SEQ ID NO:383),Tar2h-131-516 (SEQ ID NO:384), Tar2h-131-517 (SEQ ID NO:385),Tar2h-131-518 (SEQ ID NO:386), Tar2h-131-519 (SEQ ID NO:387),Tar2h-131-520 (SEQ ID NO:388), Tar2h-131-521 (SEQ ID NO:389),Tar2h-131-522 (SEQ ID NO:390), Tar2h-131-523 (SEQ ID NO:391),Tar2h-131-524 (SEQ ID NO:392), Tar21 h-131-525 (SEQ ID NO:393),Tar2h-131-526 (SEQ ID NO:394), Tar2h-131-527 (SEQ ID NO:395),Tar2h-131-528 (SEQ ID NO:396), Tar2h-131-529 (SEQ ID NO:397),Tar2h-131-530 (SEQ ID NO:398), Tar2h-131-531 (SEQ ID NO:399),Tar2h-131-532 (SEQ ID NO:400), Tar2h-131-533 (SEQ ID NO:401),TAR2h-131-534 (SEQ ID NO:402), Tar2h-131-535 (SEQ ID NO:403),Tar2h-131-536 (SEQ ID NO:404), Tar2h-131-537 (SEQ ID NO:405),Tar2h-131-538 (SEQ ID NO:406), Tar2h-131-539 (SEQ ID NO:407),Tar2h-131-539 (SEQ ID NO:408), Tar2h-131-539 (SEQ ID NO:409),Tar2h-131-540 (SEQ ID NO:410), Tar2h-131-541 (SEQ ID NO:411),Tar2h-131-542 (SEQ ID NO:412), Tar2h-131-543 (SEQ ID NO:413),Tar2h-131-544 (SEQ ID NO:414), Tar2h-131-545 (SEQ ID NO:415),Tar2h-131-546 (SEQ ID NO:416), Tar2h-131-547 (SEQ ID NO:417),Tar2h-131-548 (SEQ ID NO:418), Tar2h-131-549 (SEQ ID NO:419),Tar211-131-550 (SEQ ID NO:420), Tar2h-131-551 (SEQ ID NO:421),Tar2h-131-552 (SEQ ID NO:422), Tar2h-131-553 (SEQ ID NO:423),Tar2h-131-554 (SEQ ID NO:424), Tar2h-131-555 (SEQ ID NO:425),Tar2h-131-556 (SEQ ID NO:426), Tar2h-131-557 (SEQ ID NO:427),Tar2h-131-558 (SEQ ID NO:428), Tar2h-131-559 (SEQ ID NO:429),Tar2h-131-560 (SEQ ID NO:430), Tar2h-131-561 (SEQ ID NO:431),Tar2h-131-562 (SEQ ID NO:432), and Tar2h-131-563 (SEQ ID NO:433).

In other embodiments, the isolated and/or recombinant nucleic acidencoding a ligand that has binding specificity for TNFR1, as describedherein, wherein said nucleic acid comprises a nucleotide sequence has atleast about 80%, at least about 85%, at least about 90%, at least about91%, at least about 92%, at least about 93%, at least about 94%, atleast about 95%, at least about 96%, at least about 97%, at least about98%, or at least about 99% nucleotide sequence identity with anucleotide sequence encoding an anti-TNFR1 dAb selected from the groupconsisting of Tar2h-131 (SEQ ID NO:434), Tar2h-131 (SEQ ID NO:435),Tar2h-131-2 (SEQ ID NO:436), Tar2h-131-3(SEQ ID NO:437, Tar2h-131-4(SEQID NO:438), Tar2h-131-5(SEQ ID NO:439), Tar2h-131-6(SEQ ID NO:440),Tar2h-131-7(SEQ ID NO:441), Tar2h-131-8(SEQ ID NO:442), Tar2h-131-9(SEQID NO:443), Tar2h-131-10(SEQ ID NO:444), Tar2h-131-1 (SEQ ID NO:445),Tar2h-131-12(SEQ ID NO:446), Tar2h-131-13(SEQ ID NO:447),Tar2h-131-14(SEQ ID NO:448), Tar2h-131-15(SEQ ID NO:449),Tar2h-131-16(SEQ ID NO:450), Tar2h-131-17(SEQ ID NO:451),Tar2h-131-18(SEQ ID NO:452), Tar2h-131-19(SEQ ID NO:453),Tar2h-131-20(SEQ ID NO:454), Tar2h-131-21(SEQ ID NO:455),Tar2h-131-22(SEQ ID NO:456), Tar2h-131-23(SEQ ID NO:457),Tar2h-131-24(SEQ ID NO:458). Tar2h-131-25(SEQ ID NO:459),Tar2h-131-26(SEQ ID NO:460), Tar2h-131-27(SEQ ID NO:461),Tar2h-131-28(SEQ ID NO:462), Tar2h-131-29(SEQ ID NO:463),Tar2h-131-30(SEQ ID NO:464), Tar2h-131-31(SEQ ID NO:465),Tar2h-131-32(SEQ ID NO:466), Tar2h-131-33(SEQ ID NO:467),Tar2h-131-34(SEQ ID NO:468), Tar2h-131-35(SEQ ID NO:469),Tar2h-131-36(SEQ ID NO:470), Tar2h-131-37(SEQ ID NO:471),Tar2h-131-38(SEQ ID NO:472), Tar2h-131-39(SEQ ID NO:473),Tar2h-131-40(SEQ ID NO:474), Tar2h-131-41(SEQ ID NO:475),Tar2h-131-42(SEQ ID NO:476), Tar2h-131-43(SEQ ID NO:477),Tar2h-131-44(SEQ ID NO:478), Tar2h-131-45(SEQ ID NO:479),Tar2h-131-46(SEQ ID NO:480), Tar2h-131-47(SEQ ID NO:481),Tar2h-131-48(SEQ ID NO:482), Tar2h-131-49(SEQ ID NO:483),Tar2h-131-50(SEQ ID NO:484), Tar2h-131-51(SEQ ID NO:485),Tar2h-131-52(SEQ ID NO:486), Tar2h-131-53(SEQ ID NO:487), Tar2h-131-54(SEQ ID NO:488), Tar2h-131-55 (SEQ ID NO:489), Tar2h-131-56 (SEQ IDNO:490), Tar2h-131-57 (SEQ ID NO:491), Tar2h-131-58 (SEQ ID NO:492),Tar2h-131-59 (SEQ ID NO:493), Tar2h-131-60 (SEQ ID NO:494), Tar2h-131-61(SEQ ID NO:495), Tar2h-131-62 (SEQ ID NO:496), Tar2h-131-63 (SEQ IDNO:497), Tar2h-131-64 (SEQ ID NO:498), Tar2h-131-65 (SEQ ID NO:499),Tar2h-131-66 (SEQ ID NO:500), Tar2h-131-67 (SEQ ID NO:501), Tar2h-131-68(SEQ ID NO:502), Tar2h-131-69 (SEQ ID NO:503), Tar2h-131-70 (SEQ IDNO:504), Tar2h-131-71 (SEQ ID NO:505), Tar2h-131-72 (SEQ ID NO:506),Tar2h-131-73 (SEQ ID NO:507), Tar2h-131-74 (SEQ ID NO:508), Tar2h-131-75(SEQ ID NO:509), Tar2h-131-76 (SEQ ID NO:510), Tar2h-131-77 (SEQ IDNO:511), Tar2h-131-78 (SEQ ID NO:512), Tar2h-131-79 (SEQ ID NO:513),Tar2h-131-80 (SEQ ID NO:514), Tar2h-131-81 (SEQ ID NO:515), Tar2h-131-82(SEQ ID NO:516), Tar2h-131-83(SEQ ID NO:517), Tar2h-131-86 (SEQ IDNO:518), Tar2h-131-87 (SEQ ID NO:519), Tar2h-131-88 (SEQ ID NO:520),Tar2h-131-89 (SEQ ID NO:521), Tar2h-131-90 (SEQ ID NO:522), Tar2h-131-91(SEQ ID NO:523), Tar2h-131-92 (SEQ ID NO:524), Tar2h-131-93 (SEQ IDNO:525), Tar2h-131-94 (SEQ ID NO:526), Tar2h-131-95 (SEQ ID NO:527),Tar2h-131-96 (SEQ ID NO:528), Tar2h-1.31-97 (SEQ ID NO:529),Tar2h-131-99 (SEQ ID NO:530), Tar2h-131-100(SEQ ID NO:531),Tar2h-131-1.01 (SEQ ID NO:532), Tar2h-131-102 (SEQ ID NO:533),Tar2h-131-103 (SEQ ID NO:534), Tar2h-131-104 (SEQ ID NO:535),Tar2h-131-1.05 (SEQ ID NO:536), Tar2h-131-106 (SEQ ID NO:537),Tar2h-131-107 (SEQ ID NO:538), Tar2h-131-108 (SEQ ID NO:539),Tar2h-131-109 (SEQ ID NO:540), Tar2h-131-110 (SEQ ID NO:541),Tar2h-131-117 (SEQ ID NO:542), Tar2h-131-112 (SEQ ID NO:543),Tar2h-131-113 (SEQ ID NO:544), Tar2h-131-114 (SEQ ID NO:545),Tar2h-131-115 (SEQ ID NO:546), Tar2h-131-116 (SEQ ID NO:547),Tar2h-131-117 (SEQ ID NO:548), Tar2h-131-120 (SEQ ID NO:549),Tar2h-131-121 (SEQ ID NO:550), Tar2h-131-122 (SEQ ID NO:551),Tar2h-131-123 (SEQ ID NO:552), Tar2h-131-124 (SEQ ID NO:553),Tar2h-131-125 (SEQ ID NO:554), Tar2h-131-126 (SEQ ID NO:555),Tar2h-131-127 (SEQ ID NO:556), Tar2h-131-128 (SEQ ID NO:557),Tar2h-131-129 (SEQ ID NO:558), Tar2h-131-130 (SEQ ID NO:559),Tar2h-131-131 (SEQ ID NO:560), Tar2h-131-132 (SEQ ID NO:561),Tar2h-131-136(SEQ ID NO:562), Tar2h-131-151(SEQ ID NO:563),Tar2h-131-180 (SEQ ID NO:564), Tar2h-131-181 (SEQ ID NO:565),Tar2h-131-182 (SEQ ID NO:566), Tar2h-131-183 (SEQ ID NO:567),Tar2h-131-184 (SEQ ID NO:568), Tar2h-131-185 (SEQ ID NO:469),Tar2h-131-188 (SEQ ID NO:570), Tar2h-131-189 (SEQ ID NO:571),Tar2h-131-190 (SEQ ID NO:572), Tar2h-131-191 (SEQ ID NO:573),Tar2h-131-192 (SEQ ID NO:574), Tar2h-131-193 (SEQ ID NO:575),Tar2h-131-194 (SEQ ID NO:576), Tar2h-131-195 (SEQ ID NO:577),Tar2h-131-196 (SEQ ID NO:578), Tar2h-131-197 (SEQ ID NO:579),Tar2h-131-198 (SEQ ID NO:580), Tar2h-131-500 (SEQ ID NO:581),Tar2h-131-501 (SEQ ID NO:582), Tar2h-131-502 (SEQ ID NO:583),Tar2h-131-503 (SEQ ID NO:584), Tar2h-131-504 (SEQ ID NO:585),Tar2h-131-505 (SEQ ID NO:586), Tar2h-131-506 (SEQ ID NO:587),Tar2h-131-507 (SEQ ID NO:488), Tar2h-131-508 (SEQ ID NO:489),Tar2h-131-509 (SEQ ID NO:590), Tar2h-131-510 (SEQ ID NO:591),Tar2h-131-511 (SEQ ID NO:592), Tar2h-131-512 (SEQ ID NO:593),Tar2h-131-513 (SEQ ID NO:594), Tar2h-131-514 (SEQ ID NO:595),Tar2h-131-515(SEQ ID NO:596), Tar2h-131-516(SEQ ID NO:597),Tar2h-131-517 (SEQ ID NO:598), Tar2h)-131-518 (SEQ ID NO:599),Tar2h-131-519 (SEQ ID NO:600), Tar2h-131-520 (SEQ ID NO:601),Tar2h-131-521 (SEQ ID NO:602), Tar2h-131-522 (SEQ ID NO:603),Tar2h-131-523 (SEQ ID NO:604), Tar2h-131-524 (SEQ ID NO:605),Tar2h-131-525 (SEQ ID NO:606), Tar2h-131-526 (SEQ ID NO:607),Tar2h-131-527 (SEQ ID NO:608), Tar2h-131-528(SEQ ID NO:609),Tar2h-131-529 (SEQ ID NO:610), Tar2h-131-530 (SEQ ID NO:611),Tar2h-131-531 (SEQ ID NO:612), Tar2h-131-532 (SEQ ID NO:613),Tar2h-131-533 (SEQ ID NO:614), Tar2h-131-534 (SEQ ID NO:615),Tar2h-131-535 (SEQ ID NO:616), Tar2h-131-536 (SEQ ID NO:617),Tar2h-131-537 (SEQ ID NO:618), Tar2h-131-538 (SEQ ID NO:619),Tar2h-131-539 (SEQ ID NO:620), Tar2h-131-540 (SEQ ID NO:621),Tar2h-131-541 (SEQ ID NO:622), Tar2h-131-542 (SEQ ID NO:623),Tar2h-131-543 (SEQ ID NO:624), Tar2h-131-544 (SEQ ID NO:625),Tar2h-131-545 (SEQ ID NO:626), Tar2h-131-546 (SEQ ID NO:627),Tar2h-131-547 (SEQ ID NO:628), Tar2h-131-548 (SEQ ID NO:629),Tar2h-131-549 (SEQ ID NO:630), Tar2h-131-550(SEQ ID NO:631),Tar2h-131-551 (SEQ ID NO:632), Tar2h-131-552 (SEQ ID NO:633),Tar2h-131-553 (SEQ ID NO:634), Tar2h-131-554 (SEQ ID NO:635),Tar2h-131-555 (SEQ ID NO:636), Tar2h-131-556 (SEQ ID NO:637),Tar2h-131-557 (SEQ ID NO:638), Tar2h-131-558 (SEQ ID NO:639),Tar2h-131-559 (SEQ ID NO:640), Tar2h-131-560 (SEQ ID NO:641),Tar2h-131-561 (SEQ ID NO:642), Tar2h-131-562 (SEQ ID NO:643),Tar2m-15-2(SEQ ID NO:645), Tar2m-15-5(SEQ ID NO:646), Tar2m-15-6(SEQ IDNO:647), Tar2m-15-8(SEQ ID NO:648), Tar2m-15-9(SEQ ID NO:649), andTar2m-15-12(SEQ ID NO:650). Preferably, nucleotide sequence identity isdetermined over the whole length of the nucleotide sequence that encodesthe selected anti-TNFR1 dAb.

The invention also provides a vector comprising a recombinant nucleicacid molecule of the invention. In certain embodiments, the vector is anexpression vector comprising one or more expression control elements orsequences that are operably linked to the recombinant nucleic acid ofthe invention. The invention also provides a recombinant host cellcomprising a recombinant nucleic acid molecule or vector of theinvention. Suitable vectors (e.g., plasmids, phagmids), expressioncontrol elements, host cells and methods for producing recombinant hostcells of the invention are well-known in the art, and examples arefurther described herein.

Suitable expression vectors can contain a number of components, forexample, an origin of replication, a selectable marker gene, one or moreexpression control elements, such as a transcription control element(e.g., promoter, enhancer, terminator) and/or one or more translationsignals, a signal sequence or leader sequence, and the like. Expressioncontrol elements and a signal sequence, if present, can be provided bythe vector or other source. For example, the transcriptional and/ortranslational control sequences of a cloned nucleic acid encoding anantibody chain can be used to direct expression.

A promoter can be provided for expression in a desired host cell.Promoters can be constitutive or inducible. For example, a promoter canbe operably linked to a nucleic acid encoding an antibody, antibodychain or portion thereof, such that it directs transcription of thenucleic acid. A variety of suitable promoters for procaryotic (e.g.,lac, tac, T3, T7 promoters for E. coli) and eucaryotic (e.g. SimianVirus 40 early or late promoter, Rous sarcoma virus long terminal repeatpromoter, cytomegalovirus promoter, adenovirus late promoter) hosts areavailable.

In addition, expression vectors typically comprise a selectable markerfor selection of host cells carrying the vector, and, in the case of areplicable expression vector, an origin of replication. Genes encodingproducts which confer antibiotic or drug resistance are commonselectable markers and may be used in procaryotic (e.g. lactamase gene(ampicillin resistance), Tet gene for tetracycline resistance) andeucaryotic cells (e.g., neomycin (G418 or geneticin), gpt (mycophenolicacid), ampicillin, or hygromycin resistance genes). Dihydrofolatereductase marker genes permit selection with methotrexate in a varietyof hosts. Genes encoding the gene product of auxotrophic markers of thehost (e.g., LEU2, URA3, HIS3) are often used as selectable markers inyeast. Use of viral (e.g., baculovirus) or phage vectors, and vectorswhich are capable of integrating into the genome of the host cell, suchas retroviral vectors, are also contemplated. Suitable expressionvectors for expression in mammalian cells and prokaryotic cells (E.coli), insect cells (Drosophila Schnieder S2 cells, Sf9) and yeast (P.methanolica, P. pastoris, S. cerevisiae) are well-known in the art.

Suitable host cells can be prokaryotic, including bacterial cells suchas E. coli, B. subtilis and/or other suitable bacteria; eukaryoticcells, such as fungal or yeast cells (e.g. Pichia pastoris, Aspergillussp., Saccharomyces cerevisiae, Schizosaccharomyces pombe, Neurosporacrassa), or other lower eukaryotic cells, and cells of higher eukaryotessuch as those from insects (e.g., Drosophila Schnieder S2 cells, Sf9insect cells (WO 94/26087 (O'Connor)), mammals (e.g., COS cells, such asCOS-1 (ATCC Accession No. CRL-1650) and COS-7 (ATCC Accession No.CRL-1651), CHO (e.g., ATCC Accession No. CRL-9096, CHO DG44 (Urlaulb, G.and Chasin, L A., Proc. Natl. Acac. Sci. USA, 77(7):4216-4220 (1980))),293 (ATCC Accession No. CRL-1573), HeLa (ATCC Accession No. CCL-2), CV1(ATCC Accession No. CCL-70), WOP (Dailey, L., et al., J. Virol.,54:739-749 (1985), 3T3, 293T (Pear, W. S., et al., Proc. Natl. Acad.Sci. U.S.A., 90:8392-8396 (1993)) NS0 cells, SP2/0, HuT 78 cells and thelike, or plants (e.g., tobacco). (See, for example, Ausubel, F. M. etal., eds. Current Protocols in Molecular Biology, Greene PublishingAssociates and John Wiley & Sons Inc. (1993).) In some embodiments, thehost cell is an isolated host cell and is not part of a multicellularorganism (e.g., plant or animal). In preferred embodiments, the hostcell is a non-human host cell.

The invention also provides a method for producing a ligand (e.g.,dual-specific ligand, multispecific ligand) of the invention, comprisingmaintaining a recombinant host cell comprising a recombinant nucleicacid of the invention under conditions suitable for expression of therecombinant nucleic acid, whereby the recombinant nucleic acid isexpressed and a ligand is produced. In some embodiments, the methodfurther comprises isolating the ligand.

Ligand Formats

Ligands and dAb monomers can be formatted as mono or multispecificantibodies or antibody fragments or into mono or multispecificnon-antibody structures. Suitable formats include, any suitablepolypeptide structure in which an antibody variable domain or one ormore of the CDRs thereof can be incorporated so as to confer bindingspecificity for antigen on the structure. A variety of suitable antibodyformats are known in the art, such as, IgG-like formats, chimericantibodies, humanized antibodies, human antibodies, single chainantibodies, bispecific antibodies, antibody heavy chains, antibody lightchains, homodimers and heterodimers of antibody heavy chains and/orlight chains, antigen-binding fragments of any of the foregoing (e.g., aFv fragment (e.g., single chain Fv (scFv), a disulfide bonded Fv), a Fabfragment, a Fab′ fragment, a F(ab′)₂ fragment), a single variable domain(e.g., V_(H), V_(L), V_(HH)), a dAb, and modified versions of any of theforegoing (e.g., modified by the covalent attachment of polyalkyleneglycol (e.g., polyethylene glycol, polypropylene glycol, polybutyleneglycol) or other suitable polymer). See, PCT/GB303/002804, filed Jun.30, 2003, which designated the United States, (WO 2004/081026) regardingPEGylated of single variable domains and dAbs, suitable methods forpreparing same, increased in vivo half life of the PEGylated singlevariable domains and dAb monomers and multimers, suitable PEGs,preferred hydrodynamic sizes of PEGs, and preferred hydrodynamic sizesof PEGylated single variable domains and dAb monomers and multimers. Theentire teaching of PCT/GB03/002804 (WO 2004/081026), including theportions referred to above, are incorporated herein by reference.

The ligand can be formatted as a dimer, trimer or polymer of the adesired dAb monomers, for example using a suitable linker such as(Gly₄Ser)_(n), where n=from 1 to 8, e.g., 2, 3, 4, 5, 6 or 7. Ifdesired, ligands, including dAb monomers, dimers and trimers, can belinked to an antibody Fc region, comprising one or both of C_(H)2 andC_(H)3 domains, and optionally a hinge region. For example, vectorsencoding ligands linked as a single nucleotide sequence to an Fc regionmay be used to prepare such polypeptides.

Ligands and dAb monomers can also be combined and/or formatted intonon-antibody multi-ligand structures to form multivalent complexes,which bind target molecules with the same antigen, thereby providingsuperior avidity. For example natural bacterial receptors such as SpAcan been used as scaffolds for the grafting of CDRs to generate ligandswhich bind specifically to one or more epitopes. Details of thisprocedure are described in U.S. Pat. No. 5,831,012. Other suitablescaffolds include those based on fibronectin and affibodies. Details ofsuitable procedures are described in WO 98/58965. Other suitablescaffolds include lipocallin- and CTLA4, as described in van den Beukenet al., J. Mol. Biol. 310:591-601 (2001), and scaffolds such as thosedescribed in WO 00/69907 (Medical Research Council), which are based forexample on the ring structure of bacterial GroEL or other chaperonepolypeptides. Protein scaffolds may be combined; for example, CDRs maybe grafted on to a CTLA4 scaffold and used together with immunoglobulinV_(H) or V_(L) domains to form a ligand. Likewise, fibronectin,lipocallin and other scaffolds may be combined

A variety of suitable methods for preparing any desired format are knownin the art. For example, antibody chains and formats (e.g., IgG-likeformats, chimeric antibodies, humanized antibodies, human antibodies,single chain antibodies, bispecific antibodies, antibody heavy chains,antibody light chains, homodimers and heterodimers of antibody heavychains and/or light chains) can be prepared by expression of suitableexpression constructs and/or culture of suitable cells (e.g.,hybridomas, heterohybridomas, recombinant host cells containingrecombinant constructs encoding the format). Further, formats such asantigen-binding fragments of antibodies or antibody chains (e.g., a Fvfragment (e.g., single chain Fv (scFv), a disulfide bonded Fv), a Fabfragment, a Fab′ fragment, a F(ab′)₂ fragment), can be prepared byexpression of suitable expression constructs or by enzymatic digestionof antibodies, for example using papain or pepsin.

The ligand can be formatted as a dual specific ligand or a multispecificligand, for example as described in WO 03/002609, the entire teachingsof which are incorporated herein by reference. The dual specific ligandscomprise immunoglobulin single variable domains that have differentbinding specificities. Such dual specific ligands can comprisecombinations of heavy and light chain domains. For example, the dualspecific ligand may comprise a V_(H) domain and a V_(L) domain, whichmay be linked together in the form of an scFv (e.g., using a suitablelinker such as Gly₄Ser), or formatted into a bispecific antibody orantigen-binding fragment thereof (e.g. F(ab′)2 fragment). The dualspecific ligands do not comprise complementary V_(H)/V_(L) pairs whichform a conventional two chain antibody antigen-binding site that bindsantigen or epitope co-operatively. Instead, the dual format ligandscomprise a V_(H)/V_(L) complementary pair, wherein the V domains havedifferent binding specificities.

In addition, the dual specific ligands may comprise one or more C_(H) orC_(L) domains if desired. A hinge region domain may also be included ifdesired. Such combinations of domains may, for example, mimic naturalantibodies, such as IgG or IgM, or fragments thereof, such as Fv, scFv,Fab or F(ab′)2 molecules. Other structures, such as a single arm of anIgG molecule comprising V_(H), V_(L), C_(H)1 and C_(L) domains, areenvisaged. Preferably, the dual specific ligand of the inventioncomprises only two variable domains although several such ligands may beincorporated together into the same protein, for example two suchligands can be incorporated into an IgG or a multimeric immunoglobulin,such as IgM. Alternatively, in another embodiment a plurality of dualspecific ligands are combined to form a multimer. For example, twodifferent dual specific ligands are combined to create a tetra-specificmolecule. It will be appreciated by one skilled in the art that thelight and heavy variable regions of a dual-specific ligand producedaccording to the method of the present invention may be on the samepolypeptide chain, or alternatively, on different polypeptide chains. Inthe case that the variable regions are on different polypeptide chains,then they may be linked via a linker, generally a flexible linker (suchas a polypeptide chain), a chemical linking group, or any other methodknown in the art.

The multispecific ligand possesses more than one epitope bindingspecificity. Generally, the multi-specific ligand comprises two or moreepitope binding domains, such dAbs or non-antibody protein domaincomprising a binding site for an epitope, e.g., an affibody, an SpAdomain, an LDL receptor class A domain, an EGF domain, an avimer.Multispecific ligands can be formatted further as described herein.

In some embodiments, the ligand is an IgG-like format. Such formats havethe conventional four chain structure of an IgG molecule (2 heavy chainsand two light chains), in which one or more of the variable regions(V_(H) and or V_(L)) have been replaced with a dAb or single variabledomain of a desired specificity. Preferably, each of the variableregions (2 V_(H) regions and 2 V_(L) regions) is replaced with a dAb orsingle variable domain. The dAb(s) or single variable domain(s) that areincluded in an IgG-like format can have the same specificity ordifferent specificities. In some embodiments, the IgG-like format istetravalent and can have one, two, three or four specificities. Forexample, the IgG-like format can be monospecific and comprises 4 dAbsthat have the same specificity; bispecific and comprises 3 dabs thathave the same specificity and another dAb that has a differentspecificity; bispecific and comprise two dAbs that have the samespecificity and two dAbs that have a common but different specificity;trispecific and comprises first and second dAbs that have the samespecificity, a third dAbs with a different specificity and a fourth dAbwith a different specificity from the first, second and third dAbs; ortetraspecific and comprise four dAbs that each have a differentspecificity. Antigen-binding fragments of IgG-like formats (e.g., Fab,F(ab′)₂, Fab′, Fv, scFv) can be prepared. Preferably, the IgG-likeformats or antigen-binding fragments thereof do not crosslink TNFR1.

Half-Life Extended Formats

All antagonist of TNFR1 (e.g., ligand, dAb monomer, dimer or multimer,dual specific format, multi-specific format) can be formatted to extendits in vivo serum half life. Increased in vivo half-life is useful in invivo applications of immunoglobulins, especially antibodies and mostespecially antibody fragments of small size such as dAbs. Such fragments(Fvs, disulphide bonded Fvs, Fabs, scFvs, dAbs) are rapidly cleared fromthe body, which can severely limit clinical applications.

An antagonist of TNFR1 can be formatted to have a larger hydrodynamicsize, for example, by attachment of a polyalkyleneglycol group (e.g.polyethyleneglycol (PEG) group), serum albumin, transferrin, transferrinreceptor or at least the transferrin-binding portion thereof, anantibody Fc region, or by conjugation to an antibody domain. In someembodiments, the antagonist (e.g., ligand, dAb monomer) is PEGylated.Preferably the PEGylated antagonist (e.g., ligand, dAb monomer) bindsTNFR1 with substantially the same affinity as the same ligand that isnot PEGylated. For example, the ligand can be a PEGylated dAb monomerthat binds, wherein the PEGylated dAb monomer binds TNFR1 with anaffinity that differs from the affinity of dAb in unPEGylated form by nomore than a factor of about 1000, preferably no more than a factor ofabout 100, more preferably no more than a factor of about 10, or withaffinity substantially unchanged affinity relative to the unPEGylatedform.

Small antagonists, such as a dAb monomer, can be formatted as a largerantigen-binding fragment of an antibody or as and antibody (e.g.,formatted as a Fab, Fab′, F(ab)₂, F(ab′)₂, IgG, scFv). The hydrodynamicsize of an antagonist (e.g., ligand, dAb monomer) and its serumhalf-life can also be increased by conjugating or linking the antagonistto a binding domain (e.g., antibody or antibody fragment) that binds anantigen or epitope that increases half-live in vivo, as describedherein. For example, the antagonists (e.g., ligand, dAb monomer) can beconjugated or linked to an anti-serum albumin or anti-neonatal Fcreceptor antibody or antibody fragment, eg an anti-SA or anti-neonatalFe receptor dAb, Fab, Fab′ or scFv, or to an anti-SA affibody oranti-neonatal Fc receptor affibody.

Examples of suitable albumin, albumin fragments or albumin variants foruse in a TNFR1-binding ligand according to the invention are describedin WO 2005/077042A2, which is incorporated herein by reference in itsentirety. In particular, the following albumin, albumin fragments oralbumin variants can be used in the present invention:

-   -   SEQ ID NO:1 (as disclosed in WO 2005/077042A2, this sequence        being explicitly incorporated into the present disclosure by        reference);    -   Albumin fragment or variant comprising or consisting of amino        acids 1-387 of SEQ ID NO:1 in WO 2005/077042A2;    -   Albumin, or fragment or variant thereof, comprising an amino        acid sequence selected from the group consisting of: (a) amino        acids 54 to 61 of SEQ ID NO:1 in WO 2005/077042A2; (b) amino        acids 76 to 89 of SEQ ID NO:1 in WO 2005/077042A2; (c) amino        acids 92 to 100 of SEQ ID NO:1 in WO 2005/077042A2; (d) amino        acids 170 to 176 of SEQ ID NO:1 in WO 2005/077042A2; (e) amino        acids 247 to 252 of SEQ ID NO:1 in WO 2005/077042A2; (f) amino        acids 266 to 277 of SEQ ID NO:1 in WO 2005/077042A2; (g) amino        acids 280 to 288 of SEQ ID NO:1 in WO 2005/077042A2; (h) amino        acids 362 to 368 of SEQ ID NO:1 in WO 2005/077042A2; (i) amino        acids 439 to 447 of SEQ ID NO:1 in WO 2005/077042A2(j) amino        acids 462 to 475 of SEQ ID NO:1 in WO 2005/077042A2; (k) amino        acids 478 to 486 of SEQ ID NO:1 in WO 2005/077042A2; and (l)        amino acids 560 to 566 of SEQ ID NO:1 in WO 2005/077042A2.

Further examples of suitable albumin, fragments and analogs for use in aTNFR1-binding ligand according to the invention are described in WO03/076567A2, which is incorporated herein by reference in its entirety.In particular, the following albumin, fragments or variants can be usedin the present invention:

-   -   Human serum albumin as described in WO 03/076567A2, eg, in FIG.        3 (this sequence information being explicitly incorporated into        the present disclosure by reference);    -   Human serum albumin (HA) consisting of a single non-glycosylated        polypeptide chain of 585 amino acids with a formula molecular        weight of 66,500 (See, Meloun, et al., FEBS Letters 58.136        (1975); Behrens, et al., Fed. Proc. 34:591 (1975); Lawn, et al.,        Nucleic Acids Research 9:6102-6114 (1981); Minghetti, et al, J.        Biol. Chem. 261:6747 (1986));    -   A polymorphic variant or analog or fragment of albumin as        described in Weitkamp, et al., Ann. Hum. Genet. 37:219 (1973);    -   An albumin fragment or variant as described in EP 322094, eg,        HA(1-373, HA(1-388), HA(1-389), HA(1-369), and HA(1-419) and        fragments between 1-369 and 1-419;    -   An albumin fragment or variant as described in EP 399666, eg,        HA(1-177) and HA(1-200) and fragments between HA(1-X), where X        is any number from 178 to 199.

Where a (one or more) half-life extending moiety (eg, albumin,transferrin and fragments and analogues thereof) is used in theTNFR1-binding ligands of the invention, it can be conjugated using anysuitable method, such as, by direct fusion to the TNFR1-binding moiety(eg, anti-TNFR1 dAb or antibody fragment), for example by using a singlenucleotide construct that encodes a fusion protein, wherein the fusionprotein is encoded as a single polypeptide chain with the half-lifeextending moiety located N- or C-terminally to the TNFR1 binding moiety.Alternatively, conjugation can be achieved by using a peptide linkerbetween moieties, eg, a peptide linker as described in WO 03/076567A2 orWO 2004/003019 (these linker disclosures being incorporated by referencein the present disclosure to provide examples for use in the presentinvention).

Typically, a polypeptide that enhances serum half-life in vivo is apolypeptide which occurs naturally in vivo and which resists degradationor removal by endogenous mechanisms which remove unwanted material fromthe organism (e.g., human). For example, a polypeptide that enhancesserum half-life in vivo can be selected from proteins from theextracellular matrix, proteins found in blood, proteins found at theblood brain barrier or in neural tissue, proteins localized to thekidney, liver, lung, heart, skin or bone, stress proteins,disease-specific proteins, or proteins involved in Fc transport.

Suitable polypeptides that enhance serum half-life in vivo include, forexample, transferrin receptor specific ligand-neuropharmaceutical agentfusion proteins (see U.S. Pat. No. 5,977,307, the teachings of which areincorporated herein by reference), brain capillary endothelial cellreceptor, transferrin, transferrin receptor (e.g., soluble transferrinreceptor), insulin, insulin-like growth factor 1 (IGF 1) receptor,insulin-like growth factor 2 (IGF 2) receptor, insulin receptor, bloodcoagulation factor X, α1-antitrypsin and HNF 1α. Suitable polypeptidesthat enhance serum half-life also include alpha-1 glycoprotein(orosomucoid; AAG), alpha-1 antichymotrypsin (ACT), alpha-1microglobulin (protein HC; AIM), antithrombin III (AT III),apolipoprotein A-1 (Apo A-1), apolipoprotein B (Apo B), ceruloplasmin(Cp), complement component C3 (C3), complement component C4 (C4), C1esterase inhibitor (C1 INH), C-reactive protein (CRP), ferritin (FER),hemopexin (HPX), lipoprotein(a) (Lp(a)), mannose-binding protein (MBP),myoglobin (Myo), prealbumin (transthyretin; PAL), retinol-bindingprotein (RBP), and rheumatoid factor (RF).

Suitable proteins from the extracellular matrix include, for example,collagens, laminins, integrins and fibronectin. Collagens are the majorproteins of the extracellular matrix. About 15 types of collagenmolecules are currently known, found in different parts of the body,e.g. type I collagen (accounting for 90% of body collagen) found inbone, skin, tendon, ligaments, cornea, internal organs or type IIcollagen found in cartilage, vertebral disc, notochord, and vitreoushumor of the eye.

Suitable proteins from the blood include, for example, plasma proteins(e.g., fibrin, α-2 macroglobulin, serum albumin, fibrinogen (e.g.,fibrinogen A, fibrinogen B), serum amyloid protein A, haptoglobin,profilin, ubiquitin, uteroglobulin and β-2-microglobulin), enzymes andenzyme inhibitors (e.g. plasminogen, lysozyme, cystatin C,alpha-1-antitrypsin and pancreatic trypsin inhibitor), proteins of theimmune system, such as immunoglobulin proteins (e.g., IgA, IgD, IgE,IgG, IgM, immunoglobulin light chains (kappa/lambda)), transportproteins (e.g., retinol binding protein, α-1 microglobulin), defensins(e.g., beta-defensin 1, neutrophil defensin 1, neutrophil defensin 2 andneutrophil defensin 3) and the like.

Suitable proteins from the blood brain barrier or in neural tissueinclude, for example, melanocortin receptor, myelin, ascorbatetransporter and the like.

Suitable polypeptides that enhances serum half-life in vivo also includeproteins localized to the kidney (e.g., polycystin, type IV collagen,organic anion transporter K1, Heymann's antigen), proteins localized tothe liver (e.g., alcohol dehydrogenase, G250), proteins localized to thelung (e.g., secretory component, which binds IgA), proteins localized tothe heart (e.g., HSP 27, which is associated with dilatedcardiomyopathy), proteins localized to the skill (e.g., keratin), bonespecific proteins such as morphogenic proteins (BMPs), which are asubset of the transforming growth factor β superfamily of proteins thatdemonstrate osteogenic activity (e.g., BMP-2, BMP-4, BMP-5, BMP-6,BMP-7, BMP-8), tumor specific proteins (e.g., trophoblast antigen,herceptin receptor, oestrogen receptor, cathepsins (e.g., cathepsin B,which can be found in liver and spleen)).

Suitable disease-specific proteins include, for example, antigensexpressed only on activated T-cells, including LAG-3 (lymphocyteactivation gene), osteoprotegerin ligand (OPGL; see Nature 402, 304-309(1999)), OX40 (a member of the TNF receptor family, expressed onactivated T cells and specifically up-regulated in human T cell leukemiavirus type-I (HTLV-I)-producing cells; see Immunol. 165 (1):263-70(2000)). Suitable disease-specific proteins also include, for example,metalloproteases (associated with arthritis/cancers) including CG6512Drosophila, human paraplegin, human FtsH, human AFG3L2, murine ftsH; andangiogenic growth factors, including acidic fibroblast growth factor(FGF-1), basic fibroblast growth factor (FGF-2), vascular endothelialgrowth factor/vascular permeability factor (VEGF/VPF), transforminggrowth factor-α (TGF α), tumor necrosis factor-alpha (TNF-α),angiogenin, interleukin-3 (IL-3), interleukin-8 (IL-8), platelet-derivedendothelial growth factor (PD-ECGF), placental growth factor (P1GF),midkine platelet-derived growth factor-BB (PDGF), and fractalkine.

Suitable polypeptides that enhance serum half-life in vivo also includestress proteins such as heat shock proteins (HSPs). HSPs are normallyfound intracellularly. When they are found extracellularly, it is anindicator that a cell has died and spilled out its contents. Thisunprogrammed cell death (necrosis) occurs when as a result of trauma,disease or injury, extracellular HSPs trigger a response from the immunesystem. Binding to extracellular HSP can result in localizing thecompositions of the invention to a disease site.

Suitable proteins involved in Fc transport include, for example,Brambell receptor (also known as FcRB). This Fc receptor has twofunctions, both of which are potentially useful for delivery. Thefunctions are (1) transport of IgG from mother to child across theplacenta (2) protection of IgG from degradation thereby prolonging itsserum half-life. It is thought that the receptor recycles IgG fromendosomes. (See, Holliger et al, Nat Biotechnol 15(7):632-6 (1997).)

Methods for pharmacokinetic analysis and determination of ligandhalf-life will be familiar to those skilled in the art. Details may befound in Kenneth, A et al: Chemical Stability of Pharmaceuticals: AHandbook for Pharmacists and in Peters et al, Pharmacokinetic analysis:A Practical Approach (1996). Reference is also made to“Pharmacokinetics”, M Gibaldi & D Perron, published by Marcel Dekker,2^(nd) Rev. ex edition (1982), which describes pharmacokineticparameters such as t alpha and t beta half lives and area under thecurve (AUC).

Preparation of Immunoglobulin Based Ligands

Binding agents, antagonists, ligands, dAbs as described herein accordingto the invention can be prepared according to previously establishedtechniques, used in the field of antibody engineering, for thepreparation of scFv, “phage” antibodies and other engineered antibodymolecules. Techniques for the preparation of antibodies are for exampledescribed in the following reviews and the references cited therein:Winter & Milstein, (1991) Nature 349:293-299; Pluckthun (1992)Immunological Reviews 130:151-188; Wright et al., (1992) Crit. Rev.Immunol. 12:125-168; Holliger, P. & Winter, G. (1993) Curr. Op.Biotechn. 4, 446-449; Carter, et al. (1995) J. Hematother. 4, 463-470;Chester, K. A. & Hawkins, R. E. (1995) Trends Biotechn. 13, 294-300;Hoogenboom, H. R. (1997) Nature Biotechnol. 15, 125-126; Fearon, D.(1997) Nature Biotechnol. 15, 618-619; Plückthun, A. & Pack, P. (1997)Immunotechnology 3, 83-105; Carter, P. & Merchant, A. M. (1997) Curr.Opin. Biotechnol. 8, 449-454; Holliger, P. & Winter, G. (1997) CancerImmunol. Immunother. 45, 128-130.

Suitable techniques employed for selection of antibody variable domainswith a desired specificity employ libraries and selection procedureswhich are known in the art. Natural libraries (Marks et al. (1991) J.Mol. Biol., 222: 581; Vaughan et al. (1996) Nature Biotech., 14: 309)which use rearranged V genes harvested from human 1 cells are well knownto those skilled in the art. Synthetic libraries (Hoogenboom & Winter(1992) J. Mol. Biol., 227: 381; Barbas et al. (1992) Proc. Natl. Acad.Sci. USA, 89: 4457; Nissim et al. (1994) EMBO J, 13: 692; Griffiths etal. (1994) EMBO J., 13: 3245; De Kruif et al (1995) J. Mol. Biol., 248:97) are prepared by cloning immunoglobulin V genes, usually using PCR.Errors in the PCR process can lead to a high degree of randomisation.V_(H) and/or V_(L) libraries may be selected against target antigens orepitopes separately, in which case single domain binding is directlyselected for, or together.

Library Vector Systems

A variety of selection systems are known in the art which are suitablefor use in the present invention. Examples of such systems are describedbelow.

Bacteriophage lambda expression systems may be screened directly asbacteriophage plaques or as colonies of lysogens, both as previouslydescribed (Huse et al. (1989) Science, 246: 1275; Caton and Koprowski(1990) Proc. Natl. Acad. Sci. U.S.A., 87; Mullinax et al. (1990) Proc.Natl. Acad. Sci. U.S.A., 87: 8095; Persson et al. (1991) Proc. Natl.Acad. Sci. U.S.A., 88: 2432) and are of use in the invention. Whilstsuch expression systems can be used to screen up to 10⁶ differentmembers of a library, they are not really suited to screening of largernumbers (greater than 10⁶ members). Of particular use in theconstruction of libraries are selection display systems, which enable anucleic acid to be linked to the polypeptide it expresses. As usedherein, a selection display system is a system that permits theselection, by suitable display means, of the individual members of thelibrary by binding the generic and/or target ligands.

Selection protocols for isolating desired members of large libraries areknown in the art, as typified by phage display techniques. Such systems,in which diverse peptide sequences are displayed on the surface offilamentous bacteriophage (Scott and Smith (1990) Science, 249: 386),have proven useful for creating libraries of antibody fragments (and thenucleotide sequences that encoding them) for the in vitro selection andamplification of specific antibody fragments that bind a target antigen(McCafferty et al., WO 92/01047). The nucleotide sequences encoding thevariable regions are linked to gene fragments which encode leadersignals that direct them to the periplasmic space of E. coli and as aresult the resultant antibody fragments are displayed on the surface ofthe bacteriophage, typically as fusions to bacteriophage coat proteins(e.g., pIII or pVIII). Alternatively, antibody fragments are displayedexternally on lambda phage capsids (phagebodies). An advantage ofphage-based display systems is that, because they are biologicalsystems, selected library members can be amplified simply by growing thephage containing the selected library member in bacterial cells.Furthermore, since the nucleotide sequence that encode the polypeptidelibrary member is contained on a phage or phagemid vector, sequencing,expression and subsequent genetic manipulation is relativelystraightforward.

Methods for the construction of bacteriophage antibody display librariesand lambda phage expression libraries are well known in the art(McCafferty et al. (1990) Nature, 348: 552; Kang et al. (1991) Proc.Natl. Acad. Sci. U.S.A., 88: 4363; Clackson et al. (1991) Nature, 352:624; Lowman et al. (1991) Biochemistry, 30: 10832; Burton et al. (1991)Proc. Natl. Acad. Sci. U.S.A., 88: 10134; Hoogenboom et al. (1991)Nucleic Acids Res., 19: 4133; Chang et al. (1991) J. Immunol., 147:3610; Breitling et al. (1991) Gene, 104: 147; Marks et al. (1991) supra;Barbas et al. (1992) supra; Hawkins and Winter (1992) J. Immunol., 22:867; Marks et al., 1992, J. Biol. Chem., 267: 16007; Lerner et al.(1992) Science, 258: 1313, incorporated herein by reference).

One particularly advantageous approach has been the use of scFvphage-libraries (Huston et al., 1988, Proc. Natl. Acad. Sci. U.S.A., 85:5879-5883; Chaudhary et al. (1990) Proc. Natl. Acad. Sci. U.S.A., 87:1066-1070; McCafferty et al. (1990) supra; Clackson et al. (1991)Nature, 352: 624; Marks et al. (1991) J. Mol. Biol., 222: 581; Chiswellet al. (1992) Trends Biotech., 10: 80; Marks et al. (1992) J. Biol.Chem., 267). Various embodiments of scFv libraries displayed onbacteriophage coat proteins have been described. Refinements of phagedisplay approaches are also known, for example as described inWO96/06213 and WO92/01047 (Medical Research Council et al.) andWO97/08320 (Morphosys), which are incorporated herein by reference.

Other systems for generating libraries of polypeptides involve the useof cell-free enzymatic machinery for the in vitro synthesis of thelibrary members. In one method, RNA molecules are selected by alternaterounds of selection against a target ligand and PCR amplification (Tuerkand Gold (1990) Science, 249: 505; Ellington and Szostak (1990) Nature,346: 818). A similar technique may be used to identify DNA sequenceswhich bind a predetermined human transcription factor (Thiesen and Bach(1990) Nucleic Acids Res., 18: 3203; Beaudry and Joyce (1992) Science,257: 635; WO92/05258 and WO92/14843). In a similar way, in vitrotranslation can be used to synthesise polypeptides as a method forgenerating large libraries. These methods which generally comprisestabilised polysome complexes, are described further in WO88/08453,WO90/05785, WO90/07003, WO91/02076, WO91/05058, and WO92/02536.Alternative display systems which are not phage-based, such as thosedisclosed in WO95/22625 and WO95/11922 (Affymax) use the polysomes todisplay polypeptides for selection.

A still further category of techniques involves the selection ofrepertoires in artificial compartments, which allow the linkage of agene with its gene product. For example, a selection system in whichnucleic acids encoding desirable gene products may be selected inmicrocapsules formed by water-in-oil emulsions is described inWO99/02671, WO00/40712 and Tawfik & Griffiths (1998) Nature Biotechnol16(7), 652-6. Genetic elements encoding a gene product having a desiredactivity are compartmentalised into microcapsules and then transcribedand/or translated to produce their respective gene products (RNA orprotein) within the microcapsules. Genetic elements which produce geneproduct having desired activity are subsequently sorted. This approachselects gene products of interest by detecting the desired activity by avariety of means.

Library Construction

Libraries intended for selection, may be constricted using techniquesknown in the art, for example as set forth above, or may be purchasedfrom commercial sources. Libraries which are useful in the presentinvention are described, for example, in WO99/20749. Once a vectorsystem is chosen and one or more nucleic acid sequences encodingpolypeptides of interest are cloned into the library vector, one maygenerate diversity within the cloned molecules by undertakingmutagenesis prior to expression; alternatively, the encoded proteins maybe expressed and selected, as described above, before mutagenesis andadditional rounds of selection are performed. Mutagenesis of nucleicacid sequences encoding structurally optimised polypeptides is carriedout by standard molecular methods. Of particular use is the polymerasechain reaction, or PCR, (Mullis and Faloona (1987) Methods Enzymol.,155: 335, herein incorporated by reference). PCR, which uses multiplecycles of DNA replication catalysed by a thermostable, DNA-dependent DNApolymerase to amplify the target sequence of interest, is well known inthe art. The construction of various antibody libraries has beendiscussed in Winter et al. (1994) Ann. Rev. Immunology 12, 433-55, andreferences cited therein.

PCR is performed using template DNA (at least 1 fg; more usefully,1-1000 ng) and at least 25 pmol of oligonucleotide primers; it may beadvantageous to use a larger amount of primer when the primer pool isheavily heterogeneous, as each sequence is represented by only a smallfraction of the molecules of the pool, and amounts become limiting inthe later amplification cycles. A typical reaction mixture includes: 2μl of DNA, 25 pmol of oligonucleotide primer, 2.5 μl of 10×PCR buffer 1(Perkin-Elmer, Foster City, Calif.), 0.4 μl of 1.25 μM dNTP, 0.15 μl (or2.5 units) of Taq DNA polymerase (Perkin Elmer, Foster City, Calif.) anddeionized water to a total volume of 25 μl. Mineral oil is overlaid andthe PCR is performed using a programmable thermal cycler. The length andtemperature of each step of a PCR cycle, as well as the number ofcycles, is adjusted in accordance to the stringency requirements ineffect. Annealing temperature and timing are determined both by theefficiency with which a primer is expected to anneal to a template andthe degree of mismatch that is to be tolerated; obviously, when nucleicacid molecules are simultaneously amplified and mutagenised, mismatch isrequired, at least in the first round of synthesis. The ability tooptimise the stringency of primer annealing conditions is well withinthe knowledge of one of moderate skill in the art. An annealingtemperature of between 30° C. and 72° C. is used. Initial denaturationof the template molecules normally occurs at between 92° C. and 99° C.for 4 minutes, followed by 20-40 cycles consisting of denaturation(94-99° C. for 15 seconds to 1 minute), annealing (temperaturedetermined as discussed above; 1-2 minutes), and extension (72° C. for1-5 minutes, depending on the length of the amplified product). Finalextension is generally for 4 minutes at 72° C., and may be followed byan indefinite (0-24 hour) step at 4° C.

Combining Single Variable Domains

Domains useful in the invention, once selected, may be combined by avariety of methods known in the art, including covalent and non-covalentmethods. Preferred methods include the use of polypeptide linkers, asdescribed, for example, in connection with scFv molecules (Bird et al.,(1988) Science 242:423-426). Discussion of suitable linkers is providedin Bird et al. Science 242, 423-426; Hudson et al, Journal ImmunolMethods 231 (1999) 177-189; Hudson et al, Proc Nat Acad Sci USA 85,5879-5883. Linkers are preferably flexible, allowing the two singledomains to interact. One linker example is a (Gly₄ Ser)_(n), linker,where n=1 to 8, eg, 2, 3, 4, 5 or 7. The linkers used in diabodies,which are less flexible, may also be employed (Holliger et al., (1993)PNAS (USA) 90:6444-6448). In one embodiment, the linker employed is notan immunoglobulin hinge region.

Variable domains may be combined using methods other than linkers. Forexample, the use of disulphide bridges, provided throughnaturally-occurring or engineered cysteine residues, may be exploited tostabilise V_(H)-V_(H), V_(L)-V_(L) or V_(H)-V_(L) dimers (Reiter et al.,(1994) Protein Eng. 7:697-704) or by remodeling the interface betweenthe variable domains to improve the “fit” and thus the stability ofinteraction (Ridgeway et al., (1996) Protein Eng. 7:617-621; Zhu et al,(1997) Protein Science 6:781-788). Other techniques for joining orstabilising variable domains of immunoglobulins, and in particularantibody V_(H) domains, may be employed as appropriate.

Characterisation of Ligands

The binding of a ligand (e.g., dAb monomer, dual-specific ligand) to itsspecific antigen(s) or epitope(s) can be tested by methods which will befamiliar to those skilled, in the art and include ELISA. In a preferredembodiment of the invention binding is tested using monoclonal phageELISA. Phage ELISA may be performed according to any suitable procedure:an exemplary protocol is set forth below.

Populations of phage produced at each round of selection can be screenedfor binding by ELISA to the selected antigen or epitope, to identify“polyclonal.” phage antibodies. Phage from single infected bacterialcolonies from these populations can then be screened by ELISA toidentify “monoclonal” phage antibodies. It is also desirable to screensoluble antibody fragments for binding to antigen or epitope, and thiscan also be undertaken by ELISA using reagents, for example, against aC- or N-terminal tag (see for example Winter et al. (1994) Ann. Rev.Immunology 12, 433-55 and references cited therein.

The diversity of the selected phage monoclonal antibodies may also beassessed by gel electrophoresis of PCR products (Marks et al. 1991,supra; Nissim et al. 1994 supra), probing (Tomlinson et al., 1992) J.Mol. Biol. 227, 776) or by sequencing of the vector DNA.

Structure of Ligands

In the case that the variable domains are selected from V-generepertoires selected for instance using phage display technology asherein described, then these variable domains comprise a universalframework region, such that is they may be recognised by a specificgeneric ligand as herein defined. The use of universal frameworks,generic ligands and the like is described in WO99/20749.

Where V-gene repertoires are used variation in polypeptide sequence ispreferably located within the structural loops of the variable domains.The polypeptide sequences of either variable domain may be altered byDNA shuffling or by mutation in order to enhance the interaction of eachvariable domain with its complementary pair. DNA shuffling is known inthe art and taught, for example, by Stemmer, 1994, Nature 370: 389-391and U.S. Pat. No. 6,297,053, both of which are incorporated herein byreference. Other methods of mutagenesis are well known to those of skillin the art.

In general, nucleic acid molecules and vector constructs required forselection, preparation and formatting ligands may be constructed andmanipulated as set forth in standard laboratory manuals, such asSambrook et al. (1989) Molecular Cloning: A Laboratory Manual, ColdSpring Harbor, USA.

The manipulation of nucleic acids useful in the present invention istypically carried out in recombinant vectors.

As used herein, vector refers to a discrete element that is used tointroduce heterologous DNA into cells for the expression and/orreplication thereof. Methods by which to select or construct and,subsequently, use such vectors are well known to one of ordinary skillin the art. Numerous vectors are publicly available, including bacterialplasmids, bacteriophage, artificial chromosomes and episomal vectors.Such vectors may be used for simple cloning and mutagenesis;alternatively gene expression vector is employed. A vector of useaccording to the invention may be selected to accommodate a polypeptidecoding sequence of a desired size, typically from 0.25 kilobase (kb) to40 kb or more in length A suitable host cell is transformed with thevector after in vitro cloning manipulations. Each vector containsvarious functional components, which generally include a cloning (or“polylinker”) site, an origin of replication and at least one selectablemarker gene. If given vector is an expression vector, it additionallypossesses one or more of the following: enhancer element, promoter,transcription termination and signal sequences, each positioned in thevicinity of the cloning site, such that they are operatively linked tothe gene encoding a ligand according to the invention.

Both cloning and expression vectors generally contain nucleic acidsequences that enable the vector to replicate in one or more selectedhost cells. Typically in cloning vectors, this sequence is one thatenables the vector to replicate independently of the host chromosomalDNA and includes origins of replication or autonomously replicatingsequences. Such sequences are well known for a variety of bacteria,yeast and viruses. The origin of replication from the plasmid pBR322 issuitable for most Gram-negative bacteria, the 2 micron plasmid origin issuitable for yeast, and various viral origins (e.g. SV 40, adenovirus)are useful for cloning vectors in mammalian cells. Generally, the originof replication is not needed for mammalian expression vectors unlessthese are used in mammalian cells able to replicate high levels of DNA,such as COS cells.

Advantageously, a cloning or expression vector may contain a selectiongene also referred to as selectable marker. This gene encodes a proteinnecessary for the survival or growth of transformed host cells grown ina selective culture medium. Host cells not transformed with the vectorcontaining the selection gene will therefore not survive in the culturemedium. Typical selection genes encode proteins that confer resistanceto antibiotics and other toxins, e.g. ampicillin, neomycin, methotrexateor tetracycline, complement auxotrophic deficiencies, or supply criticalnutrients not available in the growth media.

Since the replication of vectors encoding a ligand according to thepresent invention is most conveniently performed in E. coli, an E.coli-selectable marker, for example, the β-lactamase gene that confersresistance to the antibiotic ampicillin, is of use. These can beobtained from E. coli plasmids, such as pBR322 or a pUC plasmid such aspUC18 or pUC19.

Expression vectors usually contain a promoter that is recognised by thehost organism and is operably linked to the coding sequence of interest.Such a promoter may be inducible or constitutive. The term “operablylinked” refers to a juxtaposition wherein the components described arein a relationship permitting them to function in their intended manner.A control sequence “operably linked” to a coding sequence is ligated insuch a way that expression of the coding sequence is achieved underconditions compatible with the control sequences.

Promoters suitable for use with prokaryotic hosts include, for example,the β-lactamase and lactose promoter systems, alkaline phosphatase, thetryptophan (trp) promoter system and hybrid promoters such as the tacpromoter. Promoters for use in bacterial systems will also generallycontain a Shine-Delgarno sequence operably linked to the codingsequence.

The preferred vectors are expression vectors that enables the expressionof a nucleotide sequence corresponding to a polypeptide library member.Thus, selection with the first and/or second antigen or epitope can beperformed by separate propagation and expression of a single cloneexpressing the polypeptide library member or by use of any selectiondisplay system. As described above, the preferred selection displaysystem is bacteriophage display. Thus, phage or phagemid vectors may beused, eg pIT1 or pIT2. Leader sequences useful in the invention includepelB, stII, ompA, phoA, bla and pelA. One example are phagemid vectorswhich have an E. coli. origin of replication (for double strandedreplication) and also a phage origin of replication (for production ofsingle-stranded DNA). The manipulation and expression of such vectors iswell known in the alt (Hoogenboom and Winter (1992) supra; Nissim et al.(1994) supra). Briefly, the vector contains a β-lactamase gene to conferselectivity on the phagemid and a lac promoter upstream of a expressioncassette that consists (N to C terminal) of a pelB leader sequence(which directs the expressed polypeptide to the periplasmic space), amultiple cloning site (for cloning the nucleotide version of the librarymember), optionally, one or more peptide tag (for detection),optionally, one or more TAG stop codon and the phage protein pill. Thus,using various suppressor and non-suppressor strains of E. coli and withthe addition of glucose, iso-propyl thio-β-D-galactoside (IPTG) or ahelper phage, such as VCS M13, the vector is able to replicate as aplasmid with no expression, produce large quantities of the polypeptidelibrary member only or produce phage, some of which contain at least onecopy of the polypeptide-pIII fusion on their surface.

Construction of vectors encoding ligands according to the inventionemploys conventional ligation techniques. Isolated vectors or DNAfragments are cleaved, tailored, and religated in the form desired togenerate the required vector. If desired, analysis to confirm that thecorrect sequences are present in the constricted vector can be performedin a known fashion. Suitable methods for constricting expressionvectors, preparing in vitro transcripts, introducing DNA into hostcells, and performing analyses for assessing expression and function areknown to those skilled in the art. The presence of a gene sequence in asample is detected, or its amplification and/or expression quantified byconventional methods, such as Southern or Northern analysis, Westernblotting, dot blotting of DNA, RNA or protein, in situ hybridisation,immunocytochemistry or sequence analysis of nucleic acid or proteinmolecules. Those skilled in the art will readily envisage how thesemethods may be modified, if desired.

Skeletons

Skeletons may be based on immunoglobulin molecules or may benon-immunoglobulin in origin as set forth above. Preferredimmunoglobulin skeletons as herein defined includes any one or more ofthose selected from the following: an immunoglobulin molecule comprisingat least (i) the CL (kappa or lambda subclass) domain of an antibody; or(ii) the CH1 domain of an antibody heavy chain; an immunoglobin moleculecomprising the CH1 and CH2 domains of an antibody heavy chain; animmunoglobulin molecule comprising the CH1, CH2 and CH3 domains of anantibody heavy chain; or any of the subset (ii) in conjunction with theCL (kappa or lambda subclass) domain of an antibody. A hinge regiondomain may also be included. Such combinations of domains may, forexample, mimic natural antibodies, such as IgG or IgM, or fragmentsthereof, such as Fv, scFv, Fab or F(ab′)₂ molecules. Those skilled inthe art will be aware that this list is not intended to be exhaustive.

Protein Scaffolds

Each epitope binding domain comprises a protein scaffold and one or moreCDRs which are involved in the specific interaction of the domain withone or more epitopes. Advantageously, an epitope binding domainaccording to the present invention comprises three CDRs. Suitableprotein scaffolds include any of those selected from the groupconsisting of the following: those based on immunoglobulin domains,those based on fibronectin, those based on affibodies, those based onCTLA4, those based on chaperones such as GroEL, those based onlipocallin and those based on the bacterial Fc receptors SpA and SpD.Those skilled in the art will appreciate that this list is not intendedto be exhaustive.

Scaffolds for Use in Constructing Ligands

Selection of the Main-Chain Conformation

The members of the immunoglobulin superfamily all share a similar foldfor their polypeptide chain. For example, although antibodies are highlydiverse in terms of their primary sequence, comparison of sequences andcrystallographic structures has revealed that, contrary to expectation,five of the six antigen binding loops of antibodies (H1, H2, L1, L2, L3)adopt a limited member of main-chain conformations, or canonicalstructures (Chothia and Lesk (1987) J. Mol. Biol., 196: 901; Chothia etal. (1989) Nature, 342: 877). Analysis of loop lengths and key residueshas therefore enabled prediction of the main-chain conformations of H1,H2, L1, L2 and L3 found in the majority of human antibodies (Chothia etal. (1992) J. Mol. Biol., 227: 799; Tomlinson et al. (1995) EMBO J., 14:4628; Williams et al. (1996) J. Mol. Biol., 264: 220). Although the H3region is much more diverse in terms of sequence, length and structure(due to the use of D segments), it also forms a limited number ofmain-chain conformations for short loop lengths which depend on thelength and the presence of particular residues, or types of residue, atkey positions in the loop and the antibody framework (Martin et al.(1996) J. Mol. Biol., 263: 800; Shirai et al. (1996) FEBS Letters, 399:1).

Libraries of ligands and/or domains can be designed in which certainloop lengths and key residues have been chosen to ensure that themain-chain conformation of the members is known. Advantageously, theseare real conformations of immunoglobulin superfamily molecules found innature, to minimise the chances that they are nonfunctional, asdiscussed above. Germline V gene segments serve as one suitable basicframework for constructing antibody or T-cell receptor libraries; othersequences are also of use. Variations may occur at a low frequency, suchthat a small number of functional members may possess an alteredmain-chain conformation, which does not affect its function.

Canonical stricture theory is also of use to assess the number ofdifferent main-chain conformations encoded by ligands, to predict themain-chain conformation based on ligand sequences and to choose residuesfor diversification which do not affect the canonical structure. It isknown that, in the human. V_(κ) domain, the L1 loop can adopt one offour canonical structures, the L2 loop has a single canonical structureand that 90% of human V_(κ) domains adopt one of four or five canonicalstructures for the L3 loop (Tomlinson et al. (1995) supra); thus, in theV_(κ) domain alone, different canonical structures can combine to createa range of different main-chain conformations. Given that the V_(λ)domain encodes a different range of canonical structures for the L1, L2and L3 loops and that V_(κ) and V_(λ) domains can pair with any V_(H)domain which can encode several canonical structures for the H1 and H2loops, the number of canonical structure combinations observed for thesefive loops is very large. This implies that the generation of diversityin the main-chain conformation may be essential for the production of awide range of binding specificities. However, by constructing anantibody library based on a single known main-chain conformation it hasbeen found, contrary to expectation, that diversity in the main-chainconformation is not required to generate sufficient diversity to targetsubstantially all antigens. Even more surprisingly, the singlemain-chain conformation need not be a consensus structure—a singlenaturally occurring conformation can be used as the basis for an entirelibrary. Thus, in a preferred aspect, the dual-specific ligands of theinvention possess a single known main-chain conformation.

The single main-chain conformation that is chosen is preferablycommonplace among molecules of the immunoglobulin superfamily type inquestion. A conformation is commonplace when a significant number ofnaturally occurring molecules are observed to adopt it. Accordingly, ina preferred aspect of the invention, the natural occurrence of thedifferent main-chain conformations for each binding loop of animmunoglobulin domain are considered separately and then a naturallyoccurring variable domain is chosen which possesses the desiredcombination of main-chain conformations for the different loops. If noneis available, the nearest equivalent may be chosen. It is preferablethat the desired combination of main-chain conformations for thedifferent loops is created by selecting germline gene segments whichencode the desired main-chain conformations. It is more preferable, thatthe selected germline gene segments are frequently expressed in nature,and most preferable that they are the most frequently expressed of allnatural germline gene segments.

In designing ligands (e.g., dAbs) or libraries thereof the incidence ofthe different main-chain conformations for each of the six antigenbinding loops may be considered separately. For H1, H2, L1, L2 and L3, agiven conformation that is adopted by between 20% and 100% of theantigen binding loops of naturally occurring molecules is chosen.Typically, its observed incidence is above 35% (i.e. between 35% and100%) and, ideally, above 50% or even above 65%. Since the vast majorityof H3 loops do not have canonical structures, it is preferable to selecta main-chain conformation which is commonplace among those loops whichdo display canonical structures. For each of the loops, the conformationwhich is observed most often in the natural repertoire is thereforeselected. In human antibodies, the most popular canonical structures(CS) for each loop are as follows: H1-CS 1 (79% of the expressedrepertoire), H2-CS 3 (46%), L1-CS 2 of V_(κ) (39%), L2-CS 1 (100%),L3-CS 1 of V_(κ) (36%) (calculation assumes a κ:λ ratio of 70:30, Hoodet al. (1967) Cold Spring Harbor Stomp. Quant. Biol., 48: 133). For H3loops that have canonical structures, a CDR3 length (Kabat et al. (1991)Sequences of proteins of immunological interest, U.S. Department ofHealth and Human Services) of seven residues with a salt-bridge fromresidue 94 to residue 101 appears to be the most common. There are atleast 16 human antibody sequences in the EMBL data library with therequired H3 length and key residues to form this conformation and atleast two crystallographic structures in the protein data bank which canbe used as a basis for antibody modelling (2cgr and 1tet). The mostfrequently expressed germline gene segments that this combination ofcanonical structures are the V_(H) segment 3-23 (DP-47), the J_(H)segment JH4b, the V_(κ) segment O2/O12 (DPK9) and the J_(κ) segmentJ_(κ)1. V_(H) segments DP45 and DP38 are also suitable. These segmentscan therefore be used in combination as a basis to construct a librarywith the desired single main-chain conformation.

Alternatively, instead of choosing the single main-chain conformationbased on the natural occurrence of the different main-chainconformations for each of the binding loops in isolation, the naturaloccurrence of combinations of main-chain conformations is used as thebasis for choosing the single main-chain conformation. In the case ofantibodies, for example, the natural occurrence of canonical structurecombinations for any two, three, four, five or for all six of theantigen binding loops can be determined. Here, it is preferable that thechosen conformation is commonplace in naturally occurring antibodies andmost preferable that it observed most frequently in the naturalrepertoire. Thus, in human antibodies, for example, when naturalcombinations of the five antigen binding loops, H1, H2, L1, L2 and L3,are considered, the most frequent combination of canonical structures isdetermined and then combined with the most popular conformation for theH3 loop, as a basis for choosing the single main-chain conformation.

Diversification of the Canonical Sequence

Having selected several known main-chain conformations or, preferably asingle known main-chain conformation, ligands (e.g., dAbs) or librariesfor use in the invention can be constructed by varying the binding siteof the molecule in order to generate a repertoire with structural and/orfunctional diversity. This means that variants are generated such thatthey possess sufficient diversity in their structure and/or in theirfunction so that they are capable of providing a range of activities.

The desired diversity is typically generated by varying the selectedmolecule at one or more positions. The positions to be changed can bechosen at random or are preferably selected. The variation can then beachieved either by randomisation, during which the resident amino acidis replaced by any amino acid or analogue thereof, natural or synthetic,producing a very large number of variants or by replacing the residentamino acid with one or more of a defined subset of amino acids,producing a more limited number of variants.

Various methods have been reported for introducing such diversity.Error-prone PCR (Hawkins et al. (1992) J. Mol. Biol., 226: 889),chemical mutagenesis (Deng et al. (1994) J. Biol. Chem., 269: 9533) orbacterial mutator strains (Low et al. (1996) J. Mol. Biol., 260: 359)can be used to introduce random mutations into the genes that encode themolecule. Methods for mutating selected positions are also well known inthe art and include the use of mismatched oligonucleotides or degenerateoligonucleotides, with or without the use of PCR. For example, severalsynthetic antibody libraries have been created by targeting mutations tothe antigen binding loops. The H3 region of a human tetanustoxoid-binding Fab has been randomised to create a range of new bindingspecificities (Barbas et al. (1992) Proc. Nat. Acad. Sci. USA, 89:4457). Random or semi-random H3 and L3 regions have been appended togermline V gene segments to produce large libraries with unmutatedframework regions (Hoogenboom & Winter (1992) J. Mol. Biol., 227: 381;Barbas et al. (1992) Proc. Natl. Acad. Sci. USA, 89: 4457; Nissim et al.(1994) EMBO J., 13: 692; Griffiths et al. (1994) EMBO J., 13: 3245; DeKruif et al. (1995) J. Mol. Biol., 248: 97). Such diversification hasbeen extended to include some or all of the other antigen binding loops(Crameri et al. (1996) Nature Med., 2: 100; Riechmann et al. (1995)Bio/Technology, 13: 475; Morphosys, WO97/08320, supra).

Since loop randomisation has the potential to create approximately morethan 10¹⁵ structures for H3 alone and a similarly large number ofvariants for the other five loops, it is not feasible using currenttransformation technology or even by using cell free systems to producea library representing all possible combinations. For example, in one ofthe largest libraries constructed to date, 6×10¹⁰ different antibodies,which is only a fraction of the potential diversity for a library ofthis design, were generated (Griffiths et al. (1994) supra).

Preferably, only the residues which are directly involved in creating ormodifying the desired function of the molecule are diversified. For manymolecules, the function will be to bind a target and therefore diversityshould be concentrated in the target binding site, while avoidingchanging residues which are crucial to the overall packing of themolecule or to maintaining the chosen main-chain conformation.

Diversification of the Canonical Sequence as it Applies to AntibodyDomains

In the case of antibody based ligands (e.g., dAbs), the binding site forthe target is most often the antigen binding site. Thus, preferably onlythose residues in the antigen binding site are varied. These residuesare extremely diverse in the human antibody repertoire and are known tomake contacts in high-resolution antibody/antigen complexes. Forexample: in L2 it is known that positions 50 and 53 are diverse innaturally occurring antibodies and are observed to male contact with theantigen. In contrast, the conventional approach would have been todiversify all the residues in the corresponding ComplementarityDetermining Region (CDR1) as defined by Kabat et al. (1991, supra), someseven residues compared to the two diversified in the library for useaccording to the invention. This represents a significant improvement interms of the functional diversity required to create a range of antigenbinding specificities.

In nature, antibody diversity is the result of two processes: somaticrecombination of germline V, D and J gene segments to create a naiveprimary repertoire (so called germline and junctional diversity) andsomatic hypermutation of the resulting rearranged V genes. Analysis ofhuman antibody sequences has shown that diversity in the primaryrepertoire is focused at the centre of the antigen binding site whereassomatic hypermutation spreads diversity to regions at the periphery ofthe antigen binding site that are highly conserved in the primaryrepertoire (see Tomlinson et al. (1996) J. Mol Biol., 256: 813). Thiscomplementarity has probably evolved as an efficient strategy forsearching sequence space and, although apparently unique to antibodies,it can easily be applied to other polypeptide repertoires. The residueswhich are varied are a subset of those that form the binding site forthe target. Different (including overlapping) subsets of residues in thetarget binding site are diversified at different stages duringselection, if desired.

In the case of an antibody repertoire, an initial ‘naive’ repertoire canbe created where some, but not all, of the residues in the antigenbinding site are diversified. As used herein in this context, the term“naive” refers to antibody molecules that have no pre-determined target.These molecules resemble those which are encoded by the immunoglobulingenes of an individual who has not undergone immune diversification, asis the case with fetal and newborn individuals, whose immune systemshave not yet been challenged by a wide variety of antigenic stimuli.This repertoire is then selected against a range of antigens orepitopes. If required, further diversity can then be introduced outsidethe region diversified in the initial repertoire. This maturedrepertoire can be selected for modified function, specificity oraffinity.

Naive repertoires of binding domains for the constriction of ligands inwhich some or all of the residues in the antigen binding site are variedare known in the art. (See, WO 2004/058821, WO 2004/003019, and WO03/002609). The “primary” library mimics the natural primary repertoire,with diversity restricted to residues at the centre of the antigenbinding site that are diverse in the germline V gene segments (germlinediversity) or diversified during the recombination process functionaldiversity). Those residues which are diversified include, but are notlimited to, H50, H152, H52a, H53, H55, H56, H58, H95, H96, H97, H98,L50, L53, L91, L92, L93, L94 and L96. In the “somatic” library,diversity is restricted to residues that are diversified during therecombination process (junctional diversity) or are highly somaticallymutated). Those residues which are diversified include, but are notlimited to: H31, H33, H135, H95, H96, H97, H98, L30, L31, L32, L34 andL96. All the residues listed above as suitable for diversification inthese libraries are known to make contacts in one or moreantibody-antigen complexes. Since in both libraries, not all of theresidues in the antigen binding site are varied, additional diversity isincorporated during selection by varying the remaining residues, if itis desired to do so. It shall be apparent to one skilled in the art thatany subset of any of these residues (or additional residues whichcomprise the antigen binding site) can be used for the initial and/orsubsequent diversification of the antigen binding site.

In the construction of libraries for use in the invention,diversification of chosen positions is typically achieved at the nucleicacid level, by altering the coding sequence which specifies the sequenceof the polypeptide such that a number of possible amino acids (all 20 ora subset thereof) can be incorporated at that position. Using the IUPACnomenclature, the most versatile codon is NNK, which encodes all aminoacids as well as the TAG stop codon. The NNK codon is preferably used inorder to introduce the required diversity. Other codons witch achievethe same ends are also of use, including the NNN codon, which leads tothe production of the additional stop codons TGA and TAA.

A feature of side-chain diversity in the antigen binding site of humanantibodies is a pronounced bias which favours certain amino acidresidues. If the amino acid composition of the ten most diversepositions in each of the V_(H), V_(κ) and V_(λ) regions are summed, morethan 76% of the side-chain diversity comes from only seven differentresidues, these being, serine (24%), tyrosine (14%), asparagine (11%),glycine (9%), alanine (7%), aspartate (6%) and threonine (6%). This biastowards hydrophilic residues and small residues which can providemain-chain flexibility probably reflects the evolution of surfaces whichare predisposed to binding a wide range of antigens or epitopes and mayhelp to explain the required promiscuity of antibodies in the primaryrepertoire.

Since it is preferable to mimic this distribution of amino acids, thedistribution of amino acids at the positions to be varied preferablymimics that seen in the antigen binding site of antibodies. Such bias inthe substitution of amino acids that permits selection of certainpolypeptides (not just antibody polypeptides) against a range of targetantigens is easily applied to any polypeptide repertoire. There arevarious methods for biasing the amino acid distribution at the positionto be varied (including the use of tri-nucleotide mutagenesis, seeWO97/08320), of which the preferred method, due to ease of synthesis, isthe use of conventional degenerate codons. By comparing the amino acidprofile encoded by all combinations of degenerate codons (with single,double, triple and quadruple degeneracy in equal ratios at eachposition) with the natural amino acid use it is possible to calculatethe most representative codon. The codons (AGT)(AGC)T, (AGT)(AGC)C and(AGT)(AGC)(CT)—that is, DVT, DVC and DVY, respectively using IUPACnomenclature—are those closest to the desired amino acid profile: theyencode 22% serine and 11% tyrosine, asparagine, glycine, alanine,aspartate, threonine and cysteine. Preferably, therefore, libraries areconstructed using either the DVT, DVC or DVY codon at each of thediversified positions.

Receptor Binding Assay

Antagonists of TNFR1 that inhibit binding of TNFα to TNFR1 can beidentified in a suitable receptor binding assay. Briefly, Maxisorpplates are incubated overnight with 30 mg/ml anti-human Fc mousemonoclonal antibody (Zymed, San Francisco, USA). The wells are washedwith phosphate buffered saline (PBS) containing 0.05% Tween-20 and thenblocked with 1% BSA in PBS before being incubated with 100 ng/mlTNFR1-Fc fusion protein (R&D Systems, Minneapolis, USA). Antagonists ofTNFR1 are mixed with TNF which added to the washed wells at a finalconcentration of 10 ng/ml. TNF binding is detected with 0.2 mg/mlbiotinylated anti-TNF antibody (HyCult biotechnology, Uben, Netherlands)followed by 1 in 500 dilution of horse radish peroxidase labelledstreptavidin (Amersham, Biosciences, UK) and then incubation with TMBsubstrate (KPL, Gaithersburg, USA). The reaction can be stopped by theaddition of HCl and the absorbance is read at 450 nm. Antagonists ofTNFR1 that inhibit binding of TNFα to TNFR1 lead to a decrease in TNFbinding and therefore a decrease in, absorbance compared with the TNFonly control.

L929 Cytotoxicity Assay

Antagonists of TNFR1 (e.g., ligands, dAb monomers) can be identified bythe ability to inhibit TNF-induced cytotoxicity in mouse L929fibroblasts (Evans, T. (2000) Molecular Biotechnology 15, 243-248).Briefly, L929 cells plated in microtitre plates are incubated overnightwith antagonist of TNFR1, 100 μg/ml TNF and 1 mg/ml actinomycin D(Sigma, Poole, UK). Then, cell viability is measured by readingabsorbance at 490 nm following an incubation with[3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium(Promega, Madison, USA). Antagonists of TNFR1 will inhibit cytotoxicityand therefore produce an increase in absorbance compared with TNF onlycontrol.

HeLa IL-8 Assay

Antagonists of TNFR1 (e.g., ligands, dAb monomers) can be identified bythe ability to inhibit TNF-induced secretion of IL-8 by human HeLa cells(method adapted from that of Akeson, L. et al (1996) Journal ofBiological Chemistry 271, 30517-30523, describing the induction of IL-8by IL-1 in HUVEC; here we look at induction by human TNF alpha and weuse HeLa cells instead of the HUVEC cell line). Briefly, HeLa cells canbe plated in microtitre plates were incubated overnight with antagonistof TNFR1 and 300 pg/ml TNF. Post incubation, the supernatant isaspirated off the cells and IL-8 concentration is measured using asandwich ELISA (R&D Systems), or other suitable method. Antagonists ofTNFR1 inhibit IL-8 secretion, and less IL-8 is detected in thesupernatant compared with the TNF only control.

MRC-5 IL-8 Release Assay

Antagonists of human TNFR1 can be identified using the following MRC-5cell assay. The assay is based on the induction of IL-8 secretion by TNFin MRC-5 cells and is adapted from the method described in Alceson, L.et al. Journal of Biological Chemistry 271:30517-30523 (1996),describing the induction of IL-8 by IL-1 in HUVEC. Briefly, MRC-5 cellsare plated in microtitre plates and the plates were incubated overnightwith antagonist of TNFR1 and human TNFα (300 pg/ml). Followingincubation, the culture supernatant is aspirated and the IL-8concentration in the supernatant is measured via a sandwich ELISA (R&DSystems), or other suitable method. Antagonists of TNFR1 result in adecrease in IL-8 secretion into the supernatant compared with controlwells that are incubated with TNFα only.

EXAMPLES Example 1 Antagonist of TNFR1 Locally Administered to PulmonaryTissue is Efficacious in a Subchronic Model of COPD in C57BL/6 Mice

In this study, an antagonist of TNFR1 (anti-TNFR1 dAb monomer (TAR2m21-23)) and an antagonist of TNF (ENBREL® (etanercept; ImmunexCorporation)) were administered locally to the lung by intranasaladministration 1 hour prior to each air or tobacco smoke (TS) exposure.The effects on TS-induced changes in pulmonary inflammatory indicesinduced by 11 consecutive daily TS exposures were examined 24 hoursfollowing the final exposure. The anti-TNF compound (ENBREL®(etanercept; Immunex Corporation)) was used as a control. An orallyadministered phosphodiesterase 4 (PDE4) inhibitor (BAY 19-8004;lirimilast) was also administered 1 hour prior to and 6 hours post TSexposure in another group as a reference.

Methods

Test Substance 1: EMBREL® (etanercept; Immunex Corporation)

Test Substance 2: DOM1M (anti-TNFR1 dAb monomer (TAR2m21-23))

Test Substance 3: BAY 19-8004 (PDE4 inhibitor)

The vehicle for substances 1 and 2 was Sodium citrate pH 6.0, 100 mMNaCl. The vehicle for substance 3 was 0.5% Carboxymethylcellulose(Sigma, Product No. C-4888, Lot No. 87H0036) in water. Dose volume is 5ml/kg.

Female mice (C57BL/6) full barrier bred and certified free of specificmicroorganisms on receipt (16-20 g) (Charles River) were housed ingroups of up to 5 in individually ventilated, solid bottomed cages (IVC)with Aspen chip bedding. Environments (airflow, temperature andhumidity) within the cages were controlled by the IVC system(Techniplast).

Protocols:

No. Groups: 7 Group Size: n = 10 Dose Volume: 50 μl per mouse for groups1 to 4 and 5 ml/kg for groups 1 to 7 Treatment times: One hour prior toTS or air exposure on days 1 to 11 for groups 1 to 4 and 1 hour prior toexposure and 6 hour post exposure for groups 5 to 7

The study protocol is summarized in Table 1.

TABLE 1 Group TS/Air Dose Dosing Dosing No. Exposure Test Substancemg/kg n = Route Frequency 1 Air Vehicle Na citrate 0 10 i.n. 1 hourprior to TS 2 TS Vehicle Na citrate 0 10 i.n. or air 3 TS 1 (Enbrel ®;1.0 10 i.n. exposure on etanercept, Immunex days 1 to 11 for Corp)groups 1 to 6. 4 TS 2 (DOM1m) 1.0 10 i.n. 1 hour prior & 5 Air VehicleCMC 0 10 p.o. 6 h post for 6 TS Vehicle CMC 0 10 p.o. groups 7 to 9. 7TS 3 (BAY 19-8004) 10 10 p.o. i.n., intranasal p.o., per os (oraladministration)TS Exposure

Mice (maximum 5 per exposure chamber) were exposed to TS generated fromcigarettes (Type 1R1, supplied by University of Kentucky). Initialexposure was to 4 cigarettes one day 1, and exposure was increased to amaximum, of 6 cigarettes/day by day 6/7. Exposure thereafter to Day 11was 6 cigarettes/day. The rate of increase was regulated with regard tothe daily observed tolerance of the mice. The control group of mice wasexposed to air for an equivalent length of time on each exposure day(air exposure controls).

Health Monitoring:

Animals were weighed prior to the start of the study, on day 6 of theexposure protocol, and at the time of termination. All animals weremonitored during and after each test substance administration and TSexposure.

Terminal Procedures:

Animals were sacrificed by anaesthetic overdose (pentobarbitone Na, 100mg/kg i.p.) as follows: All groups were sacrificed 24 hours after the11^(th) and final TS exposure. Mice from all treatment groups weretreated as follows: Blood samples were taken from the sub-clavianartery, placed in a microcentrifuge tube and allowed to clot overnightat 4° C. The clot was removed and the remaining fluid was centrifuged at2900 rpm in a microcentrifuge for 6 minutes. The resulting supernatantserum was decanted and stored at −40° C. for possible PK analysis. Abronchoalveolar lavage (BAL) was performed using 0.4 ml of phosphatebuffered saline (PBS). Cells recovered from the BAL were quantified bytotal and differential cell counts, Lungs were removed, snap frozen inliquid nitrogen and stored at −80° C. for possible PK analysis.

Data Analysis

A test for normality was carried out on the data. If the test waspositive, then a preliminary analysis was carried out using a one wayanalysis of variance test (one way ANOVA) followed by a Bonferroni'smultiple comparison post test to compare control and treatment groups.If the data was not normally distributed, then a Kruskal-Wall is testfollowed by Dunn's multiple comparisons test was employed. Data wereconsidered significant when p<0.05.

Results

The control group TS/vehicle had cellular infiltrates in the lungcompared to the air/vehicle group (see FIG. 1).

The TS exposed and Test Substance 1 (DOM1, anti-TNFR1 dAb) treatedgroup, showed significantly reduced cell infiltrates in the lungcompared to the TS exposed and control treated groups (FIG. 1): 72%inhibition for total cells (p<0.001), 74% for macrophages (p<0.001), 82%for neutrophils (p<0.001), 86% for lymphocytes (p<0.05) and 55% forepithelial cells (p<0.01). An 82% reduction in eosinophils was observedbut this change was not significant due to the variability in eosinophilnumbers observed in the study as a whole.

The Test Substance 3 (PDE4 inhibitor, BAY 19-8004) treated group, showedsignificantly reduced cell infiltrates in the lung compared to thecontrol group (FIG. 1): 52% inhibition for total cells (p<0.01), 55% formacrophages (p<0.01), 55% for neutrophils (p<0.001), 61% for lymphocytes(p<0.001) and 56% for eosinophils (p<0.01). A 46% reduction inepithelial cells was observed but this change was not significant.

No significant reductions in any of the cell populations were observedin the group exposed to TS and treated with Test Substance 1 (ENBREL®(etanercept; Immunex Corporation).

Example 2 Antagonist of TNFR1 Systemically Administered is Efficaciousin a Subchronic Model of COPD in C57BL/6 Mice

In this study, an antagonist of TNFR1 (Pegylated anti-TNFR1 dAb monomer(TAR2m21-23 PEGylated to increase hydrodynamic size and in vivo serumhalf-life)) and an antagonist of TNF (ENBREL® (etanercept; ImmunexCorporation)) were administered systemically by intraperitonealadministration every 48 hours beginning 24 hours prior to the initial TSexposure. The effects on TS-induced changes in pulmonary inflammatoryindices induced by 11 consecutive daily TS exposures were examined 24 hfollowing the final exposure. The anti-TNF compound (ENBREL®(etanercept; Immunex Corporation)) was used as a control.

Methods

Test Substance 1: ENBREL® (etanercept; Immunex Corporation)

Test Substance 2: PEG DOM1M (anti-TNFR1 dAb monomer (TAR2m21-23)PEGylated with a 40 kDa polyethyleneglycol to increase hydrodynamic sizeand lengthen in vivo serum half-life).

The vehicle for both Test Substances was sterile saline, and the dosevolume for both Test Substances was 10 ml/kg.

Female mice (C57BL/6) full barrier bred and certified free of specificmicro organisms on receipt (16-20 g) (Charles River) were housed ingroups of up to 5 in individually ventilated, solid bottomed cages (IVC)with Aspen chip bedding. Environments (airflow, temperature andhumidity) within the cages were controlled by the IVC system(Techniplast).

Protocols

No. Groups: 4 Group Size: n = 10 for groups 1 to 4 Dose Volume: 10 ml/kgfor groups 1 to 4 Treatment times: Every 48 hours starting 24 h prior tothe initial TS exposure. Subsequent doses to be administered 1 hourprior to TS exposure

The study protocol is summarized in Table 2. TS exposure, healthmonitoring, terminal procedures, and data analysis were performed asdescribed in Example 1.

TABLE 2 Group TS/Air Test Substance Dose Dosing Dosing No. Exposure No.mg/kg n = Route Frequency 1 Air Vehicle 0 10 i.p. At 48 hour 2 TSVehicle 0 10 i.p. intervals starting 24 h 3 TS 1 (Enbrel ®; 10 10 i.p.prior to the etanercept, initial exposure Immunex Corp) then 1 h priorto 4 TS 2 (PEG DOM1m) 10 10 i.p. alternate exposures i.p.,intraperitonealResults

The TS exposed and Test Substance 2 (PEG DOM1m) treated group, showedsignificantly reduced cell infiltrates in the lung compared to the TSexposed and control treated groups: 60% inhibition for total cells (FIG.2), 63% for macrophages, 66% for polymorphic nuclear cells, 78% forlymphocytes and 65% for eosinophils. A 40% reduction in epithelial cellswas observed but this change was not significant.

No significant reductions in any of the cell populations were observedin group exposed to TS and treated with Test Substance 1 (ENBREL®,(etanercept; Immunex Corporation). Treatment with ENBREL® (etanercept;Immunex Corporation) even led to an increase in the number of total andPMN cells in the lung.

Example 3 Pharmacokinetics of Agent that Binds TNFR1 after LocalAdministration to Pulmonary Tissue

In this study, an agent that binds TNFR1 (anti-TNFR1 dAb monomer(TAR2m21-23)) was administered locally to the lung by intranasaladministration and pharmacokinetics of the agent were evaluated.

Methods

DOM1m (anti-TNFR1 dAb monomer (TAR2m21-23)) in 20 mM sodium citratepH6.0, 100 mM NaCl was used in the study. The diluting agent was sodiumcitrate pH6.0, 100 mM NaCl.

Protocols

All animals were administered DOM1m by intranasal administration on thesame day within 1 to 2 hours of warming the solution.

Female mice (C57BL/6) full barrier bred and certified free of specificmicroorganisms on receipt (16-20 g) (Charles River) were housed ingroups of up to 5 in individually ventilated, solid bottomed cages (IVC)with Aspen chip bedding. Environments (airflow, temperature andhumidity) within the cages were controlled by the IVC system(Techniplast).

No. Groups: 5 Group Size: 3 Dose Volume: 50 μl (25 μl/nare) Sacrificetimes: 1 hour, 2 hours, 5 hours, 8 hours and 24 hours afteradministrationThe study protocol is summarized in Table 3.

TABLE 3 Concentration Sacrifice of DOM1 in time administered (afterGroup Com- Dose solution Dosing adminis- No. pound (mg/kg) (mg/ml) Routen = tration) 1 DOM1m 1 0.4 mg/ml i.n. 3 1 hour 2 DOM1m 1 0.4 mg/ml i.n.3 2 hours 3 DOM1m 1 0.4 mg/ml i.n. 3 5 hours 4 DOM1m 1 0.4 mg/ml i.n. 38 hours 5 DOM1m 1 0.4 mg/ml i.n. 3 24 hours  i.n., intranasalHealth Monitoring

Animals were weighed prior to the start of the study. All animals weremonitored during and after each compound administration. Animals in the24 hour group were monitored at regular intervals overnight

Terminal Procedures

Animals were sacrificed by anaesthetic overdose (pentobarbitone Na, 100mg/kg i.p.). Blood was taken from the subclavian artery, placed in amicrocentrifuge tube and allowed to clot overnight at 4° C. The clot wasremoved and the remaining fluid centrifuged at 2900 rpm in amicrocentrifuge for 6 minutes. The resulting supernatants were decanted,placed in a fresh tube, frozen and stored at −40° C. prior to analysis.Bronchoalveolar lavage (BAL) was collected using 0.4 ml of phosphatebuffered saline (PBS) which was instilled and withdrawn 3 times. The BALwas centrifuged at 2700 rpm in a microcentrifuge for 6 minutes and thesupernatant was removed and stored at −40° C. prior to analysis. Thecell pellet was re-suspended in a suitable volume of PBS and a totalcell count made using a haemocytometer. Cytospin slides were prepared toallow differential cell determination. The lungs were excised, snapfrozen and stored at −80° C. prior to analysis. Using a mortar andpestle, lungs were pulverized under liquid nitrogen and dissolved inT-PER Tissue Protein Extraction Reagent (Pierce) and homogenized using40 strokes with a dounce homogenizer.

ELISA to Detect DOM1M

A 96 well MAXISORP assay plate (Nunc) was coated overnight at 4° C. with100 μl per well of mTNFR/Fc (R&D systems) at 0.5 μg/ml in PBS. Wellswere washed 3 times with 0.05% Tween/PBS and 3 times with PBS. 200 μlper well of 2% BSA in PBS was added to block the plate. Wells werewashed and then 100 μl of DOM1m standard or sample was added. Plateswere incubated for 1 hour. Wells were washed, and bound DOM1 wasdetected with chicken anti-V_(H) (1/500) followed by anti-chicken IGYHRP conjugate (115000 dilution; Abcam). Plates are developed with 100 μlof SureBlue 1-Component TMB MicroWell Peroxidase (1(KPL, Gaithersburg,USA) solution which was added to each well, and the plate was left atroom temperature until a suitable signal had developed (˜5 minutes). Thereaction was stopped by the addition of HCl and the absorbance was readat 450 nm.

Results

The level of DOM1m in the BAL was maximum at 1 hour and was about 14μg/ml (about 3.5 μg in 0.25 ml of BAL fluid). This means that at least17% (3.5 μg of 20 μg total administered) of the administered materialwas present in the bronchoalveolar compartment of the lung. Morematerial may be present in the surrounding tissues but this materialcannot be recovered. The levels in the BAL were high for a prolongedperiod of time and showed a gradual decline over 24 hours (? 10-folddecline after 24 hours).

The levels of DOM1m in the lung tissue were relatively constant up to 8hours after administration, and were undetectable 24 hours afteradministration. At 8 hours after administration the levels in the lungtissue were about 0.35 μg. The percentage of the total administered dosepresent in the lung tissue at 8 hours was about 2% (Total doseadministered was 20 μg). Taken together with the BAL levels, the maximumlevel of the agent detected in the lung as a whole at the time pointsexamined was at least ˜20% of the total dose administered.

The level of DOM1m in the serum was maximum at 1 hour (about 150 ng/ml)and rapidly declined. At 5 hours after administration, the levels in theserum were about 70 ng/ml, which is equivalent to 100 ng/mouse (1.5 mlof blood volume). The percentage of the total administered dose presentin the serum 5 hours after administration was about 0.5% (Total doseadministered was 20 μg). DOM1m was not detectable in the serum after 5hours.

Example 4 Cross Reactivity with Cynomolgus TNFR1

A cynomolgus (Macaca fascicularis) skin fibroblast cell line was used totest cross-reactivity of anti-human TNFR1 dAbs to cynomolgus TNFR1 in acell-based TNFR1 assay. Cross-reactivity to cynomolgus TNFR1 isadvantageous because pharmacokinetic and toxicology studies can beperformed without using a surrogate agent.

Method

Cynomolgus embryo skin fibroblast cells (5×10³ cells per well) wereincubated with anti-human TNFR1 dAb for 1 hour at 37° C./5% CO₂. 200pg/ml (final concentration) of human TNF was then added, and the platewas incubated overnight at 37° C./5% CO₂.

The human IL-8 DuoSet ELISA, was used to measure the concentration ofhuman IL-8 in the cell culture supernatants. The assay was carried outaccording to the manufacture's instructions. A 96 well Nunc Maxisorpassay plate was coated with 100 μl detection antibody at 4 μg/ml in PBS.The plate was incubated overnight at 4 C. In between each incubationstep the plates were washed three times with 0.05% tween/PBS and threetimes with PBS using an automated plate washer. 200 μl per well of 1%BSA/PBS was added and the plate was incubated for 1 hour at roomtemperature. 90 μl of 0.1% BSA, 0.05% Tween-20 in PBS was added to eachwell and 10 μl cell supernatant, a standard curve was included of IL-8starting at 5 ng/ml in 0.1% BSA, 0.05% Tween-20 in PBS. 100 μl ofdetection antibody was added at 20 ng/ml (stock solution diluted 1:180in 0.1% BSA, 0.05% Tween-20 in PBS) to each well and the plates wereincubated for 2 h at room temperature. 100 μl of streptavidin-HRP wasadded to each well (stock solution diluted 1:200 in 0.1% BSA, 0.05%Tween-20 in PBS). The plates were incubated for 20 mins at roomtemperature. 100 μl of SureBlue 1-Component TMB MicroWell Peroxidasesolution was added to each well and left at room temperature until theblue colour develops. The reaction was stopped by adding 100 μl 1Mhydrochloric acid. The absorbance in a plate was read at 450 nm within30 mins.

Results/Conclusions

Anti-human TNFR1 Abs in the TAR2h-131 series are able to effectivelyblock TNF induced IL-8 release by cynomolgus fibroblasts. The dAbsTAR2h-131-511 and TAR2h-131-117 had slightly higher potency values inthe cynomolgus assay (303 nM and 330 nM, respectively) as compared topotency measured in a human MRC-5 assay (˜600 pM). In conclusionTAR2h-131 series dAbs are cross reactive with cynomolgus TNFR1.

Example 5 Effects of a Pulmonary Delivered Anti-TNFR1 Dab onTNFα-Induced Pulmonary Inflammation

An anti-TNFR1 dAb was administered to the lungs of mice by theintranasal route 1 hour prior to intranasal delivery of TNFα. The effectof pre-dosing the lung with an anti-TNFR1 dAb prior to TNFαadministration was investigated by determining the number of neutrophilsin the BAL, quantifying the concentration of the inflammatory cytokinesKC, MIP-2 and MCP-1 in BAL, and quantifying E-selectin in lung tissue atselected timepoints after TNFα administration.

Methods

The inflammatory stimulus was recombinant murine TNFα in PBS containing0.1% BSA.

The anti-TNFR1 dAb was TAR2m-21-23 (Batch BH31/01/06-1) in 20 nM citratebuffer pH 6

TNFα (1 μg per mouse) was administered by the intranasal (i.n.) route.The volume administered was 50 μl per mouse (20 μg/ml). The anti-TNFR1dAbTAR2m-21-23 (1 mg/kg) was also administered by the i.n. route. Thevolume of dAb administered was 50 μl per mouse (0.4 mg/ml as mice were20 g).

Female mice (C57/bl6) full barrier bred and certified free of specificmicro organisms on receipt (16-20 g) (Charles River) were housed ingroups of up to 5 in individually ventilated, solid bottomed cages (IVC)with Aspen chip bedding. Environments (airflow, temperature andhumidity) within the cages were controlled by the IVC system(Techniplast).

Treatment Groups:

No. Groups: 13 Group Size: n = 5-6

Groups of mice were dosed i.n. with either vehicle or dAb at 1 hourprior to TNFα administration. Groups were sacrificed at predeterminedtimes after TNFα administration as listed in Table 4.

TABLE 4 Sacrifice time post agonist Group Anti-TNFR1 TNFα (hours) n AVehicle Vehicle 2-8 6 B Vehicle 1 μg/mouse 2 5 C dAb 1 mg/kg 1 μg/mouse2 5 D Vehicle 1 μg/mouse 4 5 E dAb 1 mg/kg 1 μg/mouse 4 5 F Vehicle 1μg/mouse 6 6 G dAb 1 mg/kg 1 μg/mouse 6 6 H Vehicle 1 μg/mouse 8 6 I dAb1 mg/kg 1 μg/mouse 8 6 J Vehicle 1 μg/mouse 24 5 K dAb 1 mg/kg 1μg/mouse 24 5 L Vehicle 1 μg/mouse 48 5 M dAb 1 mg/kg 1 μg/mouse 48 5

Approximately 3 minutes prior to treatment, light anaesthesia wasinduced by Isofluorane inhalation Vehicle or the dAb was instilled in avolume of 50 μl/mouse by the i.n. route. Mice were allowed to recoverand then returned to the home cage. After 1 hour TNFα (1 μg/mouse) orits vehicle were administered by the same i.n. route. Groups of dAb orvehicle treated mice were sacrificed at 2, 4, 6, 8, 24 and 48 hours postTNFα administration.

Mice were killed by anaesthetic overdose (pentobarbitone Na, 100 mg/kgi.p.). The trachea was cannulated and bronchoalveolar lavage (BAL)conducted using 3 separate 0.4 ml aliquots of PBS. The lavage fluid waskept on ice prior to centrifugation. Hearts and lungs were removed enbloc, the heart was removed and the lungs were snap frozen using liquidnitrogen. The lavage fluid was centrifuged and the supernatant dividedinto four 250 μl aliquots and stored at −40° C. The cell pellet wasre-suspended in 40 μl of PBS, and 10 μl of the cell suspension takeninto 90 μl of ‘Kimura’ or ‘Turks’ stain for total wet cell counts usinga haemocytometer. Cytospin slides were prepared from the cell suspensionand stained using ‘Wrights Giemsa’ stain for differential cell analysis.Cells were differentiated using standard morphometric techniques.

Sample preparation and ELISA: ELISA kits for murine TNFα, KC, MIP-2,MCP-1 and E-selectin were obtained from R&D Systems. Neat BALsupernatant samples were used for determination of the concentration ofthe cytokines KC, MIP-2 and MCP-1 in BAL. BAL, supernatant was diluted1:100 for determination of the concentration of TNFα in BAL.

Frozen lung was thawed and homogenized in 500 μl of lysis buffercontaining 10 mM HEPES pH 7.5, 0.5% triton X-100, 150 mM NaCl, 1 mMEDTA, 0.5 mM AEBSF and a protease inhibitor cocktail (1 μg/ml leupeptin,1 μg/ml aprotinin, 10 μg/ml trypsin-chymotrypsin inhibitor and 1 μg/mlpepstatin). Lung homogenate supernatant was diluted for determination oflung tissue E-selectin (1:50) and TNFα (1:100) concentrations.

BAL and lung homogenate supernatant total protein concentrations weredetermined using a Quantipro BCA kit (Sigma).

Results

Following intranasal administration of murine TNFα significant levels ofBAL and lung tissue TNFα were detected 2 hrs post administration (FIGS.11A and 11B). Concentrations of TNFα in both BAL and tissue decreased ina time-dependent fashion, and returned to baseline (vehicle treated)levels by 48 hrs. Levels in the BAL were approximately 10 times higherthan levels in the lung tissue. These data demonstrate that i.n.administration of TNFα resulted in significant lung delivery of thecytokine. It is possible however, that some of the TNFα detected couldbe of endogenous origin although the profile of the response does notsuggest this.

A 1 hour pre-dose of 1 mg/kg of the anti-TNFR1 dAb had no obvious effecton BAL and lung tissue TNFα concentration throughout the time pointsexamined. Despite a rapid increase in BAL and lung tissue TNFαconcentration following i.n. administration of murine TNFα, BALneutrophil numbers did not significantly increase above baseline between1 and 8 hours post TNFα administration. However, at 24 and 48 hrs postTNFα administration, BAL neutrophilia was observed (FIG. 12). Theincrease in BAL neutrophils at 24 and 48 hrs was partially inhibited bythe anti-TNFR1 dAb (26% and 44% inhibition respectively).

Concentrations of the neutrophil chemoattractants KC and MIP-2 weresignificantly increased 2 hrs following TNFα administration, anddecreased in a time-dependent fashion with both chemokines returning tobasal concentrations by 24 hrs (FIGS. 13A and 13B). These increases inBAL KC and MIP-2 were significantly reduced by anti-TNFR1 dAbpre-treatment.

Concentrations of BAL MCP-1 and lung tissue E-selectin were alsosignificantly increased by TNFα, but the peak increase was later thanthat of BAL KC and MIP-2 (6 hrs rather than 2 hrs). Concentrations ofBAL MCP-1 and lung tissue E-selectin returned to basal levels by 48 hrs(FIGS. 13C and 13D). These increases in BAL KC and MIP-2 weresignificantly reduced by anti-TNFR1 dAb pre-treatment.

Discussion

Intranasal administration of murine TNFα to mice resulted in significantlevels of pulmonary TNFα, which induced significant increases in BALneutrophils, BAL KC, MIP-2 and MCP-1 and lung tissue E-selectin. BALneutrophils were not increased until about 24 hu-s and remained elevatedat 48 hrs. Previous data in the literature using rats demonstrated aprotracted neutrophil response. The peak increases in BAL KC and MIP-2were observed at the 2 hr time point, with peak increases in BAL MCP-1and lung tissue E-selectin at around 6 hrs. Increased levels of each ofKC, MIP-2, MCP-1 and E-selecting were detected 6 hrs afteradministration of TNFα.

Pre-treatment with an anti-TNFR1 dAb (1 mg/kg) 1 hr beforeadministration of murine TNFα inhibited the increases in BAL neutrophilsand KC, MIP-2 and MCP-1 and lung tissue E-selectin. These data suggeststhe TNFR1 receptor mediates the majority of the pulmonary inflammationinduced by i.n. TNFα in this model.

Example 6 Effects of Varying Doses of Pulmonary Delivered Anti-TNFR1 dAbon TNFα-Induced Pulmonary Inflammation

Varying doses of an anti-TNFR1 dAb were administered to the lungs ofmice by the i.n. route 1 hour prior to intranasal delivery of TNFα. Thedoses of anti-TNFR1 dAb used were 2.5 mg/kg, 1 mg/kg, 0.3 mg/kg, 0.1mg/kg, 0.03 mg/kg and 0.01 mg/kg. The effect of pre-dosing with ananti-TNFR1 dAb was investigated by quantifying the concentration of theinflammatory cytokines KC, MIP-2 and MCP-1 in BAL, and quantifyingE-selectin in lung tissue.

Methods

The inflammatory stimulus and test substance were the same as thosedescribed in Example 5. TNFα (1 μg per mouse) was administered by theintranasal (i.n.) route. The volume administered was 50 μl per mouse (20μg/ml).

The anti-TNFR1 dAb TAR2m-21-23 was administered by the i.n. route at adose of 2.5 mg/kg, 1 mg/kg, 0.3 mg/kg, 0.1 mg/kg, 0.03 mg/kg or 0.01mg/kg. The volume of dAb administered was 50 μl per mouse.

Mice were of the same strain and housed as described in Example 5.

Treatment Groups:

Group Size: n=4-7

Groups of mice were dosed i.n. with either vehicle or dAb (2.5, 1, 0.3,0.1, 0.03 or 0.01 mg/kg) at 1 hour prior to TNFα administration. Allgroups were sacrificed 6 hrs after TNFα administration.

Mice were dosed using the same procedure as in Example 6. Mice werekilled 6 hrs after i.n. administration of TNFα, and BAL cells, BALsupernatant and frozen lung tissue collected as described in Example 6.BAL protein, KC, MIP-2 and MCP-1, lung homogenate supernatant proteinand E-selectin were examined and quantified as described in Example 6.

Results

As shown in Example 5, intranasal administration of TNFα inducedsignificant concentrations of KC, MIP-2 and MCP-1 in the BAL andE-selectin in the lung tissue 6 hrs after dosing. Pre-treatment (1 hrprior to TNFα) with an anti-TNFR1 dAb inhibited the elevation of theseinflammatory mediators in a dose dependent fashion (Table 5) althoughthe potency did vary between mediators.

TABLE 5 Dose dependent inhibition of TNFα-induced increases in BAL KC,MIP-2 and MCP-1 and lung tissue E-selectin concentrations.¹ dAb i.n.dosing (mg/kg) 2.5 1 0.3 0.1 0.03 0.01 BAL KC 66 ± 10 82 ± 8  71 ± 15 70± 40 77 ± 33  7 ± 48 BAL MIP-2 89 ± 16 91 ± 8  77 ± 14 74 ± 39 61 ± 3314 ± 78 BAL MCP-1 64 ± 13 60 ± 21 60 ± 9  none none none Lung E selectin59 ± 18 65 ± 10 59 ± 18 30 ± 30 none none ¹The table shows percentinhibition (mean ± SD) of TNFα-induced increases in BAL KC, MIP-2 andMCP-1 and lung tissue E-selectin concentrations at dAb doses of 2.5, 1,0.3, 0.1, 0.03 and 0.01 mg/kg.

The anti-TNFR1 dAb inhibited BAL KC to a similar degree at all dosesbetween 2.5 mg/kg and 0.03 mg/kg, and was inactive at 0.01 mg/kg.Similarly, the anti-TNFR1 dAb inhibited BAL MIP-2 to substantially thesame degree at all doses between 2.5 mg/kg and 0.1 mg/kg, with slightlyless inhibition at 0.03 mg/kg, and was inactive at 0.01 mg/kg.

The anti-TNFR1 dAb was a less potent inhibitor of BAL MCP-1 assignificant inhibition was observed between 2.5 mg/kg and 0.3 mg/kg, butnot at lower doses. Lung tissue E-selectin concentrations were inhibitedin a similar fashion; significant inhibition was observed between 2.5mg/kg and 0.3 mg/kg, minimal inhibition at 0.1 mg/kg, and no effect atlower doses.

Discussion

Intranasal administration of murine TNFα to mice induced significantincreases in BAL neutrophils, BAL KC, MIP-2 and MCP-1, and lung tissueE-selectin. These increases were significantly inhibited in adose-dependent fashion with an anti-TNFR1 dAb. The anti-TNFR1 dAb hadmore potent inhibitory activity on BAL KC and MIP-2 compared with BALMCP-1 and lung tissue E-selectin. This might be because peak increasesin BAL KC and MIP-2 were induced relatively quickly following TNFαadministration, whereas peak increases in BAL MCP-1 and tissueE-selectin were later.

This study demonstrates that i.n. anti-TNFR1 dAb a dose of 0.3 mg/kgsignificant inhibited TNFα-induced increases in BAL neutrophils, BAL KC,MIP-2 and MCP-1, and lung tissue E-selectin at six hours post TNFαadministration, but that 0.3 mg/kg is not a supra-maximal dose.

Example 8 Duration of Action In Vivo of an Anti-TNFR1 Dab Administeredby the Intranasal Route

Anti-TNFR1 dAb (TAR2m-21-23) was administered to the lungs of mice bythe i.n. route at various times prior to i.n. administration of TNFα.The effect of pre-dosing the lung with anti-TNFR1 dAbs was investigatedby quantifying the inflammatory cytokines KC, MIP-2 and MCP-1 in BAL,and quantifying E-selectin in lung tissue, by ELISA. The results areshown in Table 6.

Methods:

Inflammatory stimulus: recombinant murine TNFα

Test Substance 1: TAR2m-21-23 (Batch BH31/01/06-1) (anti-TNFR1 dAb)

Vehicle for ml TNFα PBS containing 0.1% BSA.

Vehicle for TAR2m-21-23: 20 nM citrate buffer pH 6

Dose of TNFα was 1 μg per mouse by the intranasal (i.n.) route as usedin the previous study. Volume administered to the nose was 50 g-1 permouse (20 μg/ml). Dose of TAR2m-21-23 was 0.3 mg/kg by i.n. route.Volume administered to the nose was 50 μl per mouse (0.4 mg/ml as micewere 20 g).

Mice used and dAb preparation: Mice were of the same strain and housedas detailed in the previous study protocol.

Protocols:

Treatment Groups:

Group Size: n=4-7

Treatment times:

Groups of mice were dosed i.n. with either vehicle or dAb at 1, 2, 4, or6 hours prior to TNFα administration. All groups were sacrificed 6 hrsafter TNFα administration.

Dosing and Terminal procedures: Mice were dosed as detailed above usingthe same procedure as the previous study. Mice were killed 6 hrs afteri.n. TNFα administration and BAL cells, BAL supernatant and frozen lungtissue were collected as detailed in the previous study.

Sample preparation and ELISA: BAL protein, KC, MIP-2 and MCP-1 and lunghomogenate supernatant protein and E-selectin were examined as detailedin the previous experiment.

TABLE 6 Duration of action in vivo of an anti-TNFR1 dAb.² TNF (no dAbpre-dose time (hrs) Veh dAb) 1 2 4 6 BAL KC 32 145 77 61 46 39 BAL MIP-214 54 18 18 13 8 BAL MCP-1 3 418 123 90 218 188 Lung E selectin 3 44 2023 27 28 ²The table shows mean BAL KC (pg/ml), MIP-2 (pg/ml) and MCP-1(pg/ml) and lung tissue E-selectin (ng/ml) concentrations six hoursafter i.n. administration of TNFα or vehicle. Anti-TNFR1 dAb wasadministered 1, 2, 4, or 6 hours prior to administration of TNFα.

The results presented in Table 6, show that administration of dAb at 1,2, 4, or 6 hours prior to administration of TNFα significantly inhibitedof BAL KC and MIP-2 induced by TNF at all dAb pre-dose timepoints.Better inhibition was observed at longer pre-dose times. Thissuggests >1 hr is optimal for dAb binding to the receptor following i.n.dosing. In a similar manner administration of dAb at 1, 2, 4, or 6 hoursprior to administration of TNFα also significantly inhibited BAL MCP-1and lung tissue E-selectin induced by TNF at all dAb pre-dosetimepoints. These results show that anti-TNFR1 dAb has a duration ofaction that is greater than 6 hours.

Example 9 Evaluation of an Anti-TNFR1 dAb that Binds TNFR1 and InhibitsBinding of TNFα to the Receptor Administered by the Intranasal Route onTNFα-Induced Pulmonary Inflammation.

The previous examples show that an anti-TNFR1 dAb (“non-competitive dAb”TAR2m-21-23) which binds TNFR1 but does not inhibit binding of TNFα toTNFR1 significantly inhibited TNFα-induced pulmonary inflammation. Thisstudy demonstrates that a dAb that binds TNFR1 and inhibits binding ofTN-Fα to TNFR1 (“competitive dAb” TAR-m-15-12) was also efficacious ininhibiting TNFα-induced pulmonary inflammation.

Methods:

Inflammatory stimulus: recombinant murine TNFα

Test Substance 1: TAR2m-21-23 (Batch BH31/01/06-1) (competitiveanti-TNFR1 dAb)

Test Substance 2: TAR2m-15-12 (non-competitive anti-TNFR1 dAb)

Vehicle for rm TNFα: PBS containing 0.1% BSA.

Vehicle for dAbs: 20 nM citrate buffer pH 6

Dose of TNFα was 1 μg per mouse by the intranasal (i.n.) route as usedin the previous study. Volume administered to the nose was 50 μl permouse (20 μg/ml).

Dose of TAR2m-21-23 or TAR2m-15-12 was 0.3, 0.1, and 0.03 mg/kg by i.nroute. Volume administered to the nose was 50 μl per mouse (0.4 mg/ml asmice were 20 g).

Mice used and dAb preparation: Mice were of the same strain and housedas detailed in the previous study protocol. dAb was formulated aspreviously described.

Protocols:

Treatment Groups:

Group Size: n=4-7

Treatment times:

Groups of mice were dosed i.n. with either vehicle or dAb at 1 hr priorto TNFα administration. All groups were sacrificed 6 hrs after TNFαadministration. Dosing and Terminal procedures: Mice were dosed asdetailed above using the same procedure as the previous study. Mice werekilled 6 hrs after i.n. TNFα administration and BAL cells, BALsupernatant and frozen lung tissue collected as detailed in the previousstudy.

Sample preparation and ELISA: BAL protein, KC, MIP-2 and MCP-1 and lunghomogenate supernatant protein and E-selection were examined as detailedin the previous experiment.

TABLE 7 Dose dependent effects of a competitive or a non-competitive ananti-TNFR1 dAb on TNFα-induced increases in BAL KC, MIP-2 and MCP-1 andlung tissue E-selectin concentrations.³ non-competitive competitive dAbi.n dAb i.n dosing dosing (mg/kg) (mg/kg) 0.3 0.1 0.03 0.3 0.1 0.03 BALKC  97 ± 15¹ 67 ± 22 70 ± 16 105 ± 20  66 ± 9  43 ± 22 BAL 78 ± 8  78 ±14 80 ± 14 88 ± 11 66 ± 18 54 ± 16 MIP-2 BAL 71 ± 11 34 ± 19 41 ± 8  82± 26  1 ± 60  1 ± 41 MCP-1 Lung E 60 ± 4  42 ± 34 19 ± 25 55 ± 27 13 ±8  14 ± 7  selectin ³The table show the percent inhibition (mean ± SD)of TNFα-induced increases in BAL KC, MIP-2 and MCP-1 and lung tissueE-selectin concentrations at dAb doses 0.3, 0.1, and 0.03 mg/kg.

The results present in Table 7 show that similar to previous studiespre-treatment (1 hr prior to TNFα) with a non-competitive anti-TNFR1 dAb(TAR2m-21-23) dose dependently inhibited the elevation of theinflammatory mediators BAL KC, MIP-2 and MCP-1 and lung tissueE-selectin. In a similar manner a competitive anti-TNFR1 dAb(TAR2m-15-12) also dose dependently inhibited the elevation of theinflammatory mediators BAL KC, MIP-2 BAL and MCP-1 and lung tissueE-selectin.

The TAR2m-15-12 anti-TNFR1 dAb had slightly less efficacy on BAL KC at0.03 mg/kg, and on BAL MIP-2 at 0.1-0.03 mg/kg compared withTAR2m-21-23. The TAR2m-15-12 anti-TNFR1 dAb was inactive on BAL MCP-1 at0.1 mg/kg compared with TAR2m-21-23 which showed 34% inhibition on BALMCP-1. The TAR2m-15-12 anti-TNFR1 dAb had slightly less efficacy on lungtissue E-selectin at 0.1 mg/kg compared with TAR2m-21-23.

The in vitro potency was ˜1 nM for TAR2m-21-23 and ˜5 nM forTAR2m-15-12. In addition TAR2m-21-23 has greater affinity and sloweroff-rate than TAR2m-15-12. The difference in efficacy on theinflammatory mediators is likely to be due to reduced affinity andpotency. This indicates that there is no obvious difference between anon-competitive dAb (TAR2m-21-23) and a competitive dAb TAR12m-15-12that inhibits binding of TNFα to TNFR1.

SUMMARY

Table 8 summarizes the results of TS induced studies and TNF inducedstudies. The TS exposed and PEGylated anti-TNFR1 dAb treated group (10mg/kg), showed significantly reduced cell infiltrates in the lungcompared to the TS exposed and control treated groups: 62% inhibitionfor total cells. No significant reductions in any of the cellpopulations were observed in the (10 mg/kg i.p.) TS/ENBREL® (etanercept;Immunex Corporation) treated group. Significantly reduced cellinfiltrates were only observed in the TS/ENBREL® (etanercept; ImmunexCorporation) treated group when dosing was increased to 30 mg/kg (i.p.).This indicates that >3-fold higher systemic mg/kg dosing of ENBREL®(etanercept; Immunex Corporation) is required compared to PEGylatedanti-TNFR1 dAb to achieve significant reductions in cell populations.

In the TNFα-induced model, the anti-TNFR1 dAb treated group (0.3 mg/kgi.n.), show 78% inhibition of TNFα induced MIP-2 levels. A similar levelof inhibition in MIP-2 levels (78%) was achieved in the ENBREL®(etanercept; Immunex Corporation) group treated with 10 mg/kg (i.n.).The ENBREL® (etanercept; Immunex Corporation) group treated with 1 mg/kg(i.n.) did not show any significant reductions of cell influx. Togetherthis data indicates that >30 fold higher i.n. mg/kg dosing of ENBREL®(etanercept; Immunex Corporation) is required compared to anti-TNFR1 dAbto achieve efficient inhibition of MIP-2.

TABLE 8 Potency, ND₅₀ MW, L929 Dosing Molecule kD t½_(β) assay ROA(mg/kg) Inhibition of cell influx PEG-TAR2m 52 2-4 d 1 nM i.p.  10 mg/kg62% (p < 0.001) ENBREL ® (etanercept; 150 1 d 5-50 pM i.p.  10 mg/kg −1%(ns) Immunex Corporation) (estimated) ENBREL ® (etanercept; 150 1 d 5-50pM i.p.  30 mg/kg 37% Immunex Corporation) (estimated) (p < 0.01) Ratanti-TNF mAb 150 2-7 d 6-8 nM i.p.   3 mg/kg 51% (estimated) (mTNF) (p <0.001) TAR2m monomer 12 4-6 hrs i.n. 1 nM i.n.   1 mg/kg 53%/72% (p <0.05) ENBREL ® (etanercept; 150 1 d 5-50 pM i.n.   1 mg/kg 11% (ns)Immunex Corporation) (estimated) Reduction in MIP-2 levels TAR2m 12 4-6hrs i.n. 1 nM i.n.   1 mg/kg 120% ENBREL ® (etanercept; 150 1 d 5-50 pMi.n.  10 mg/kg  78% Immunex Corporation) (estimated) Rat anti-TNF mAb150 2-7 d 6-8 nM i.n. 2.5 mg/kg  87% (estimated) (mTNF) TAR2m monomer 124-6 hrs i.n. 1 nM i.n. 0.3 mg/kg  78% TAR2m monomer 12 4-6 hrs i.n. 5 nMi.n. 0.3 mg/kg  88% (competitive dAb) TAR2m monomer 12 4-6 hrs i.n. 1 nMi.n. 0.1 mg/kg  78% TAR2m monomer 12 4-6 hrs i.n. 5 nM i.n. 0.1 mg/kg 66% (competitive dAb)

While this invention has been particularly shown and described withreferences to preferred embodiments thereof, it will be understood bythose skilled in the art that various changes in form and details may bemade therein without departing from the scope of the inventionencompassed by the appended claims.

What is claimed is:
 1. A ligand comprising a dAb that comprises a CDR1amino acid sequence, a CDR2 amino acid sequence and a CDR3 amino acidsequence of a dAb having the amino acid sequence shown in SEQ ID NO:379.
 2. A dAb that binds TNFR1, wherein the dAb is an antagonist of theTNFR1 and comprises a CDR1 amino acid sequence, a CDR2 amino acidsequence and a CDR3 amino acid sequence of the dAb having the amino acidsequence shown in SEQ ID NO:
 379. 3. A dAb comprising a CDR1 amino acidsequence, a CDR2 amino acid sequence and a CDR3 amino acid sequence ofthe dAb having the amino acid sequence shown in SEQ ID NO:
 379. 4. A dAbthat binds TNFR1, wherein the dAb is an antagonist of the TNFR1 andcomprises the amino acid sequence shown in SEQ ID NO:
 379. 5. A dAbcomprising the amino acid sequence shown in SEQ ID NO:
 379. 6. A ligandcomprising an immunoglobulin single variable domain that binds TNFR1;wherein the amino acid sequence of the immunoglobulin single variabledomain comprises CDR1, CDR2 and CDR3 amino acid sequences derived fromthe amino acid sequence shown in SEQ ID NO: 379; wherein the first threeamino proximal residues of CDR1 comprise up to two amino acidsubstitutions in CDR1 from the amino acid sequence shown in SEQ ID NO:379; wherein CDR2 has at least about 80% identity to CDR2 of the aminoacid sequence shown in SEQ ID NO: 379; and wherein CDR3 is the same asCDR3 of the amino acid sequence shown in SEQ ID NO: 379.